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Microsomal Particles and
Protein Synthesis

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Marine Biological Laboratory Library

Woods Hole, Massachusetts

Microsomal Particles and
Protein Synthesis

Papers presented at the First Symposium of the
Biophysical Society ', at the Massachusetts Institute
of Technology j Cambridge, February 5, 6, and 8, 1958

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B(ot

SYMPOSIUM

Biophysical Society

Microsomal Particles and
Protein Synthesis

RICHARD B. ROBERTS, Editor

Published on behalf
of the

WASHINGTON ACADEMY OF SCIENCES
WASHINGTON, D. C.

PERGAMON PRESS

NEW YORK • LONDON • PARIS • LOS ANGELES

1958

Library of Congress Catalog Card Number 58-13658

THE LORD BALTIMORE PRESS, INC.

FOREWORD

The formation of a new society provides an occasion for innovations in the
forms of meetings and publications. The Program Committee of the Bio-
physical Society attempted to seize this opportunity in arranging the 1958 meet-
ing at Cambridge. In addition to the usual short contributed papers, review
papers were scheduled for the three afternoon sessions to inform the member-
ship of progress and problems in selected areas of biophysics. The contributed
papers in these areas were allocated ample time for presentation and discus-
sion, resulting in what might be called "contributed symposia." Those work-
ing in the field had the advantages of a symposium type of meeting; those less
familiar had an introduction provided by the review papers followed by an
opportunity to participate in the detailed technical sessions.

It also appeared desirable to alter the usual publication procedures. Custom-
arily the complete proceedings of "invited symposia" are published but only
the abstracts of short contributions. The twenty papers dealing with micro-
somal particles normally would be published individually during the next two
years, scattered through different issues of five or ten journals. Alternatively,
it would be possible to publish a transcript of the symposium. This procedure
would provide a more complete account than is given in the abstracts; the
material would be in one volume; and it could be issued much more rapidly
than individual contributions to journals. Both the Council of the Biophysical
Society and a majority of the contributors favored publication of the symposium
material.

Transcripts, however, require an enormous editorial effort. It therefore
seemed preferable to request the contributors to provide their own edited tran-
scripts. These transcripts have been accepted with the understanding that pub-
lication in this volume which records material presented at a meeting would
not preclude later publication in the usual journals.

The purpose of this volume is not to present well established theories or
reviews of well known work. Rather, it is to publish new facts and new data
while they are still fresh, useful, and possibly wrong. The relative costs of re-
search time and publication make such a book worth purchasing if it saves
no more than an hour of research time. The entire publication costs are justi-
fied if the book can save a month in some research program.

vi MICROSOMAL PARTICLES

The Washington Academy of Sciences has agreed to act as publisher in the
hope of demonstrating that this type of book can be brought out rapidly and
that it does serve a useful function.

We wish to thank the Office of Publications of Carnegie Institution of Wash-
ington for editorial help in seeing the project through the press.

D. B. COWIE

Chairman, Monograph Committee
Washington Academy of Sciences

C. LEVINTHAL

Chairman, Program Committee
Biophysical Society

R. B. ROBERTS, Editor

Carnegie Institution of Washington
Department of Terrestrial Magnetism
5241 Broad Branch Road, Northwest
Washington 15, D. C.

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INTRODUCTIO

The topic "Microsomal Particles and Protein Synthesis" seemed particularly
appropriate for the first symposium of the Biophysical Society. The particles
owe their recognition to the electron microscope and the ultracentrifuge. X-ray
diffraction studies will undoubtedly contribute to the picture of the structure
of the particles; radioactive tracers show the kinetics of formation of the par-
ticles and their products; radiation experiments can give other evidence about
the role of the particles in protein synthesis. Thus many of the special areas
of competence of biophysicists are involved in the study of the particles.

More important, however, is the timeliness of a symposium devoted to a
discussion of these ubiquitous granules. For a number of years circumstantial
evidence has accumulated which indicates that ribonucleic acid (RNA) is
implicated in protein synthesis. More recently it has been recognized that a
large part of the RNA occurs in the form of ribonucleoprotein (RNP) par-
ticles. Particles of roughly the same size and composition have been isolated
from sources differing as widely as rat liver, pea seedlings, and microorganisms.
Accordingly there has developed a widespread faith that the particles are an
important part of the machinery for protein synthesis.

It must be admitted, however, that the evidence is entirely circumstantial. A
number of arguments would come immediately to the mind of a lawyer de-
fending the particles from the charge of protein synthesis. (1) In vivo experi-
ments have shown incorporation of radioactive tracers which is initially higher
in the microsome fraction than in the soluble proteins, but the kinetic data are
not sufficiently complete to prove a precursor-product relationship. For exam-
ple, it is possible that steady-state conditions do not prevail; there is no cer-
tainty that both components draw on the same pool of amino acids; only the
average of the soluble proteins is measured, whereas individual components
might behave quite differently. (2) There are many cases both in complete and
in cell-free systems where RNAase has been observed to inhibit protein syn-
thesis. In many of these the addition of RNA (not RNP) is sufficient to
restore the synthetic activity. (3) Cell-free systems showing unequivocal pro-
tein synthesis use cells that are only partially disrupted, and the requirement
for particles is not demonstrated. In those systems where the particles have
been partly purified, the incorporation data are more suggestive of exchange
than of true protein synthesis. (4) No mechanism has been suggested which
shows how the structure of the particle is compatible with its function as the
template for synthesis of long chains. It appears that the particles have not yet

viii MICROSOMAL PARTICLES

been proved guilty beyond all reasonable doubt. In the last few years, however,
there have been marked advances in the study of the particles which promise
to resolve these lingering doubts. Thus a symposium dealing with the particles
and their function in living cells could hardly fail to bring forth new and
exciting information.

In this symposium a number of papers were concerned with methods of
isolation, the size, the composition, and the stability of the particles. One strik-
ing observation was that particles of roughly 80 S are found in a wide variety
of materials; another area of agreement was in the requirement for magnesium
to stabilize the particles. There was a consensus of opinion that carefully puri-
fied particles have little enzymatic activity and that their RNA content is 40
per cent or more. Several reports showed that the protein moieties of nucleo-
protein have certain distinctive properties. Other studies explored the reasons
for the variations in particle sizes that are observed both in vivo and in vitro.
New kinetic data were presented which indicate that the protein of the par-
ticles does not serve as precursor material for nonparticulate protein. The in-
corporation of adenylamino acids was demonstrated in one study which also
illuminated the need for caution in the interpretation of incorporation studies.
Other papers reported less direct methods of approach to the understanding of
the particles and their role in protein synthesis, such as studies of radiation
effects and studies of incorporation of amino acid analogs. All together these
reports provide a number of new facts that must be taken into account by any
theory of protein and nucleic acid synthesis.

During the course of the symposium a semantic difficulty became apparent.
To some of the participants, microsomes mean the ribonucleoprotein particles
of the microsome fraction contaminated by other protein and lipid material;
to others, the microsomes consist of protein and lipid contaminated by particles.
The phrase "microsomal particles" does not seem adequate, and "ribonucleo-
protein particles of the microsome fraction" is much too awkward. During the
meeting the word "ribosome" was suggested; this seems a very satisfactory
name, and it has a pleasant sound. The present confusion would be eliminated
if "ribosome" were adopted to designate ribonucleoprotein particles in the size
range 20 to 100 S.

The symposium provided to the participants an opportunity for comparing
notes on methods and techniques and for exchange of views on the status of
various problems. It undoubtedly affected the immediate research plans of a
number of the participants. This volume is being published in the hope that
it will extend some of these benefits to those who did not attend.

CONTENTS

1. Isolation and Characterization of Bacterial Nucleoprotein Particles — ■
William C. Gillchriest and Robert M. Boc/{ 1

2. The Stabilization and Physical Characteristics of Purified Bacterial
Ribonucleoprotein Particles — Jac\ Wagman and Weston R. Trawic\ 11

3. Stability of Ribonucleoprotein Particles of Escherichia coli — Ellis T.
Bolton, Bill H. Hoyer, and Daniel B. Ritter 18

4. Biochemical Characterization and Electron-Microscopic Appearance

of Microsome Fractions — David Garfin^el 22

5. The Configurational Properties of Ribonucleic Acid Isolated from
Microsomal Particles of Calf Liver — Benjamin D. Hall and Paul Doty 27

6. Microsomes and Ribonucleoprotein Particles [Invited paper] — George

E. Palade . 36

7. The Influence of Conditions of Culture on Certain Soluble Macro-
molecular Components of Escherichia coli — S. Dagley and J. Sy\es . 62

8. Physicochemical and Metabolic Studies on Rat Liver Nucleoprotein —
Mary L. Peter mann, Mary G. Hamilton, M. Earl Balis, Kumud
Samarth, and Pauline Pecora 70

9. Ultracentrifugal Studies of Microsomes from Starving, Nonproliferat-
ing, and Proliferating Yeast — James K. Ashi\awa 76

10. Fractionation of Escherichia coli for Kinetic Studies — Richard B.
Roberts, Roy J. Britten, and Ellis T. Bolton 84

11. Microsomal Structure and Hemoglobin Synthesis in the Rabbit Retic-

ulocyte— Howard M. Dintzis, Henry Borsoo\, and Jerome Vinograd 95

12. Effects of /7-Fluorophenylalanine on the Growth and Physiology of
Yeast — G. N. Cohen, H. O. Halvorson, and S. Spiegelman . . . 100

13. Enzymatic and Nonenzymatic Synthesis in Adenyl Tryptophan —
Martin Karasel(, Paul Casteljranco, P. R. Krishnaswamy, and Alton
Meister 109

14. Participation of Adenyl Amino Acids in Amino Acid Incorporation
into Proteins — Paul Casteljranco, Alton Meister, and Kivie Moldave 115

75243

x MICROSOMAL PARTICLES

15. The Synthesis of Hydroxyproline within Osteoblasts [Abstract] —
Sylvia Fitton Jackson 121

16. Studies on Amino Acid Incorporation in Bacteria Using Ionizing
Radiation — Ellis Kempner and Ernest Pollard 123

17. The Effect of X Rays on the Incorporation of Phosphorus and Sulfur
into Escherichia coli — Ernest Pollard and Jane Kennedy .... 136

18. Statistical Relations in the Amino Acid Order of Escherichia coli
Protein — Harold J. Morowitz 147

19. The Formation of Protomorphs — Eran\ T. McClure and Richard B.
Roberts 151

20. Structure of Microsomal Nucleoprotein Particles from Pea Seedlings
-Paul 0. P. Ts'o 156

Isolation and Characterization
of Bacterial Nucleoprotein Particles

WILLIAM C. GILLCHRIEST ROBERT M. BOCK

Department of Biochemistry, University of Wisconsin

Fractionation of the particulate matter from broken cells has long excited
the biochemist. Lilienfeld [1] prepared nuclear and cytoplasmic fractions and
studied the properties of a deoxynucleoprotein (DNP). Huiskamp [2] noted
the influence of buffer salts on isolated DNP. The possibility of differential
extraction of subcellular structures was investigated by Bensley and Hoerr [3].
The technique of purifying subcellular components has advanced rapidly
through the efforts of Claude [4], Hogeboom and Schneider [5], and
Anderson [6].

The fractionation of subcellular components offers the possibility of integrat-
ing the fields of intracellular anatomy, cellular physiology, and biochemistry.
Siekevitz [7] working with mitochondria and Palade [8] and Zamecnik [9]
working with the microsomal fraction have begun this integration by equating
isolated fractions to structures observed in the electron microscope. Our studies
with the ribonucleoprotein of Azotobacter vinelandii have clearly demon-
strated that progress in this integration of fields demands a detailed under-
standing of the properties and stability of the subcellular particles. Previous
studies [10] on the protein synthesis in cell-free extracts of A. vinelandii must
now be reinterpreted in the light of our current understanding of the stability
of bacterial ribonucleoprotein.

In 1954 Palade and Porter [8a] demonstrated endoplasmic reticulum in ani-
mal cells. Hodge, Martin, and Morton [11] in 1957 demonstrated similar struc-
tures in plant cells, and Sacks [12] has found related structures in yeasts, higher
molds, and algae, leaving, at the present time, only the bacteria without clearly
demonstrated endoplasmic reticulum. Pochon [13] found structures in A. vine-
landii which by staining and observation in the light microscope were identi-
fied as nuclei. We have observed granularity in regions of thin sections through

2 MICROSOMAL PARTICLES

A. vinelandii examined in the electron microscope. Several speakers at this
conference have referred to similar granularity in sections of Escherichia coli as
ribonucleoprotein particles, but no clear identification of these granules as such
has yet been accomplished. Ribonucleoprotein particles, similar in size and
chemical composition to those from animal cells, yeast, and fungi [14], can,
however, be prepared from bacteria [15]. Several papers in this volume describe
the ribonucleoprotein of E. coli, and this paper will treat the preparation and
properties of ribonucleoprotein particles from A. vinelandii.

RUPTURE OF BACTERIAL CELLS

One of the aims of this work is to prepare subcellular structures having a
useful correspondence to structures that existed in the intact cell. We have no
single criterion to indicate when such a preparation has been accomplished,
but we use as supporting evidence reproducibility of the product when pre-
pared by several varying methods and we also invoke all the information about
the stability of isolated components. The stability observations are described
in a later section of this paper. Three different methods of cell rupture have
been found to permit isolation of indistinguishable particles, provided that the
cultures used were harvested at a similar stage of growth.

The first method was physical grinding with number 320 mesh Carborundum
which had been washed with hydrochloric acid and rinsed with distilled water
until neutral. The cells had been previously washed with distilled water. The
packed cell paste was ground with 4 parts by weight Carborundum for approxi-
mately 15 minutes or until moist. An additional 2 parts of Carborundum was
added, and the cells were ground for approximately 5 minutes more. Visible
microscopic examination of the mixture revealed that approximately 95 per
cent of the cells were ruptured by the grinding method. The ground cells were
diluted with 8 times the original cell volume of the following buffer: 1.6 X 10"3
M K2HPO4, 0.4 X 10"3 M KH2PO4, and 5 X 10"3 M MgS04. This will be re-
ferred to as the RNP buffer. The supernate from centrifuging this mixture at
500g for 30 minutes is referred to as the crude extract.

The second method of cell breakage employed cells grown in the presence
of 2 M glycerol. The cells were collected by low-speed centrifugation, and the
pellet was diluted into 8 volumes of the RNP buffer to rupture and produce
the crude extract.

In the third method of cell breakage, A. vinelandii protoplasts were ruptured
by osmotic shock. Weibull [16] showed that Bacillus megatherium, when
treated with lysozyme in sucrose solutions of high osmotic pressure, changed
to a spherical form which is readily ruptured by lowering the osmotic pressure.
We have avoided the use of sucrose in view of our findings on the instability
of the isolated particles in dilute sucrose solutions. The A. vinelandii cells
were washed in the RNP buffer, and suspended in 1.5 XlO"3 M EDTA at a
dilution such that the optical density at 660 m\\ was approximately 0.75. These
solutions had been previously osmotically adjusted with glycerol or with Carbo-

GILLCHRIEST AND BOCK 3

wax "4000" to maintain the protoplasts. The turbid solutions were brought to
13 ug/ml in crystalline egg white lysozyme, and the turbidity was observed until
its rapid decrease ceased. The protoplasts were then collected by low-speed
centrifugation, washed once in osmotically adjusted RNP buffer, collected
again, and ruptured by osmotic shock upon dilution with 10 times the packed
cell volume of RNP buffer to yield a crude extract. During the development
of the protoplast procedure, both the formation of the protoplasts and their
osmotic rupture upon dilution were followed in the visible and phase contrast
microscope.

PURIFICATION OF RIBONUCLEOPROTEIN PARTICLES (fig. 1)

The crude extract derived from any of the above three procedures is centri-
fuged at 4900g for 30 minutes. The pellet that accumulates consists of cell debris

PARTICLE

PREPARATION

CRUDE EXTRACT

(4,900g x 30min)

DISCARD SUPERNATE

(I05,400g x 60min)

PELLET

DISCARD

(8,700 g x 15 min)

1
DISCARD PREPARATION

Fig. 1. Flow sheet for differential centrifugation of ribonucleoprotein from a crude
extract of A. vinelandii prepared by I grinding, II osmotic shock, or III protoplastic osmotic
shock.

4 MICROSOMAL PARTICLES

and, with method I, some Carborundum that was not removed at lower speeds.
The supernatant liquid from this step is now centrifuged at 105,00% for 60
minutes. The pellet so obtained is solubilized in RNP buffer for approximately
12 hours, and then centrifuged at 8700g for 15 minutes. The precipitate is dis-
carded, and the supernatant liquid is examined in the analytical ultracentrifuge
to determine the number of sedimenting components, their relative amounts,
and their sedimentation coefficients. When the 86 S component is desired, the
105,000^- and the 8700^ cycle is repeated until over 90 per cent of the area in the
schlieren pattern is under the appropriate peak.

PROPERTIES OF RIBONUCLEOPROTEIN PARTICLES

When a crude extract is processed to the stage labeled "preparation" in
figure 1, and is examined in the analytical ultracentrifuge, it is found to sedi-
ment as a single peak of sedimentation coefficient 86 S. If, however, the same
crude extract is carried to the same stage employing a buffer in which the mag-
nesium concentration has been reduced to 10~3 M, the ultracentrifuge pattern
now shows five significant components. Comparison of the schlieren and ultra-
violet absorption photographs in the ultracentrifuge suggests that all these com-
ponents contain nucleoprotein. The sedimentation coefficients extrapolated to
zero concentration and corrected to 20° C are 86, 77, 58, 39, and 10 S. The 86,
58, and 39 S components are usually found in largest amount. Figures 2 and 3

Fig. 2. An electron micrograph of the edge of a droplet of RNP particles sprayed onto
a collodion membrane and shadowed with uranium. Magnified 34,000 times. Taken on a
Siemens Elmiskop I by Professor Paul Kaesbcrg.

GILLCHR1EST AND BOCK

Fig. 3. An electron micrograph of a central portion of a sprayed droplet showing RNP
particles magnified 170,000 times. The smallest particles are about 200 A in diameter, the
largest about 250 A. From the shapes of their shadows it is estimated that their thicknesses
are about 75 per cent as great as their diameters. Taken on a Siemens Elmiskop I by
Professor Paul Kaesberg.

are electron micrographs of a purified preparation of the 86 S particles which
was diluted lOOOfold with distilled water and then quickly sprayed on a col-
lodion membrane and air-dried. It is not yet known how the short exposure to
distilled water will affect particle structure. The electron micrographs taken
under these conditions show that at least two size classes are present, both of
which are roughly spherical. The 86, 58, and 39 S particles all show small de-
pendence of sedimentation coefficient on concentration, which also suggests
that the particles are not markedly asymmetric.

The particles appear to contain ribonucleic acid and protein and to be free
of lipid and deoxy nucleic acid. The nucleic acid component has been sepa-
rated and purified by detergent treatment [17], phenol [18], chloroform [19],
and glacial acetic acid [20] extraction. The protein component when sepa-
rated from the nucleic acid has been found to be insoluble in aqueous solutions
unless prepared through a 67 per cent glacial acetic acid procedure. The pro-
tein shows an ultraviolet absorption typical of a protein rich in tyrosine. We
have derived only one major protein from the particle at this point. The de-
rived protein appears to have only one type of N-terminal amino acid, which
we have very tentatively identified as glycine. The number of protein subunits
per particle has not yet been quantitatively determined, but the experiments to
date suggest a large number.

6 MICROSOMAL PARTICLES

Ribose nucleic acid prepared from the particle by chloroform extraction of
the protein shows markedly the hyperchromic effect characteristic of poly-
merized nucleic acids. Immediately upon addition of the alkali the optical
density at 260 mu increases 15 per cent. After incubation for 16 hours at 30° C
with 0.5 N NaOH and adjusting the pH to 7.5, the final hyperchromic effect
is found to be 39.1 per cent. The nucleotides (table 1) arising upon alkaline
hydrolysis of the ribose nucleic acid have been chromatographed on Dowex-1-
formate ion-exchange columns developed with gradient elution. The unknown
nucleotide shows chromatographic behavior similar to that of the new ribonu-
cleotide reported by Cohn, but its acid, alkaline, and neutral ultraviolet ab-
sorption spectra are not identical to those of the fifth nucleotide which we have
isolated from yeast.

The 86 S particle has been examined for its stability as a function of salt,
chelating agents, enzymatic attack, pH, and sucrose concentration (fig. 4) . The
results of these studies were fed back into improvements in the preparative pro-
cedure and are of utmost importance to the interpretation of labeled amino
acid incorporation studies in the particulate fractions of A. vinelandii. If the
86 S particle is suspended in a pH 7.05 buffer of 2xl0"3 M phosphate, 10~3
M MgSO-i, with NaCl added to a total ionic strength of 0.03, or is dialyzed
against 2 X 10"3 M K2HPC>4:KH2P04 (4:1) buffer, it dissociates to yield 58 and
39 S components.

Our early studies showed that the 58 and 39 S components could be returned
to 10^3 M Mg++-containing solutions without re-forming the 86 S particle which
had previously been stable to that environment. Encouraged by our discussions
with Dr. Paul Ts'o at this conference, we explored further and found that in
5 X 10"3 M Mg++ the 58 and 39 S components recombined to form the 86 S par-
ticle, and that once formed this particle again was stable in 10"3 Mg++. In all
these studies the buffer also contained 2 X 10"3 M potassium phosphate buffer
of pH 7.05.1 We also confirm Ts'o's observation that the area of the 39 S peak
is about one-half that of the 58 S peak, which suggests that one small 39 S
and one larger 58 S particle combine to form the 86 S particle. Upon addition
of 0.01 M, pH. 7, ethylenediaminetetraacetic acid, the particles further dissociated
to ribonucleoprotein of sedimentation coefficient less than 5 S (not extrapolated
to zero concentration). The particles are also rapidly degraded to small frag-
ments by ribonuclease but are not attacked by deoxyribonuclease. They are
precipitated by pH below 6.5 or above 7.5.

Attempts to use sucrose for certain stages of the purification led to the ob-
servation that, if sucrose was added to the RNP buffer, the particles aggregated
and were readily removed by low-speed centrifugation. Sucrose concentrations
from 3 to 30 per cent were all found to have this effect. This finding necessi-

1 Note added in proof: We recently reported at the 1958 meeting of the Federation of
American Societies for Experimental Biology that a buffer 5xl0~3 M in MgO and ad-
justed to p¥L 7.05 with cacodylic acid gives improved yield and excellent stability of the
80 S class of RNP from yeast, E. coli, and A. vinelandii.

GILLCHR1EST AND BOCK

TABLE 1. Analysis of Nucleic Acid Hydrolyzed with 0.5 AT NaOH

for 16 Hours at 37° C

Hydrolyzate was chromatographed on a Dowex-1-formate column
with gradient elution.

Nucleoprotein

Whole

Particle,

Cells,*

mole %

AMP

GMP

CMP

UMP

Unknown

nucleotide

Not reported

* Whole cell data from

Lombard and

Chargaff [1

!1].

tates re-evaluation of some studies [10] that have been carried out on A. vine-
landii and raises the important question whether this phenomenon can occur
in ribonucleoprotein from other sources.

The ribonucleoprotein has been assayed for a large number of enzymatic
activities. It appears free of nucleotide phosphatase activity, glucose-1-phos-
phatase, oxidative phosphorylation enzymes, and electron-transport enzymes.
A feeble glucose-6-phosphatase activity of 10 mM phosphate released per minute

4900g SUPERNATE

t — J

STANDARD

SALT

RNASE

-i

EDTA

DNASE

Fig. 4. The preparation and stability of A. vinelandii nucleoprotein. Each analytical
ultracentrifuge pattern is labeled with the stage of preparation (top pair) or the treatment
to which a pure 86 S product was subjected. The salt and EDTA treatment are described
in the text. The RNAse action occurred in less than 5 minutes at 1 part of enzyme per
1000 parts of particle whereas the DNAse at the same concentration produced no change
in 45 minutes.

8 MICROSOMAL PARTICLES

per gram of particle was detected, but it is at most a few per cent of that found
in equal weights of liver microsome fractions.

DISCUSSION

The study of A. vinelandii ribonucleoprotein has been more successful in
posing interesting questions than in providing answers to previous questions.
Can the sharp requirements of the nucleoprotein for divalent cations and for
certain pW ranges be exploited to give information on the mode of combina-
tion of nucleic acid and protein or combination between nucleoprotein subunits ?
Does the marked difference in ribonuclease sensitivity of the plant ribonucleo-
protein viruses and the bacterial ribonucleoprotein imply a different orientation
or localization of nucleic acid and protein? The plant viruses now appear to
have a protein coating with nucleic acid (or nucleoprotein) in an inner layer
concentric with the protein coat and thus protected from ribonuclease attack.
How different must the structure be to permit the rapid attack observed?

Will the small subunits derivable by salt and EDTA treatment also yield in-
formation on the mode of action or size of the functional nucleic acid?

The striking effect of divalent cations on the physical state of nucleoprotein
is now becoming recognized as a phenomenon common to many systems.
Huiskamp [2] in 1901 noted that thymus nucleoprotein was precipitated by
0.01 M calcium, barium, and magnesium salts and dissolved in excesses (0.1 M)
of these same salts. He equated changes in physical properties of nucleoprotein
solutions during dialysis to losses of divalent cations. He noted that heavy-
metal divalent cations formed nucleoprotein precipitates that were difficult to
dissolve. Korkes [22] and co-workers used a similar observation on manganese
RNP to remove RNP from bacterial extracts. Carter and Hall [23] working
in the laboratories of J. W. Williams noted that thymus nucleoprotein in
sodium chloride solutions was a rodlike molecule but in calcium chloride solu-
tions it became compact and showed no dependence of sedimentation rate on
concentration. Wiberg and Neuman [24] have studied the binding of mag-
nesium and calcium by RNA and DNA and find a region of concentration
through which the number of equivalents bound changes rapidly. This con-
centration range is the same as that which we find critical for nucleoprotein
structural changes.

The studies reported here, added to the work of Mazia [25] on the role of
polyvalent cations in deoxynucleoprotein and nuclear structure and of Chao
and Schachman [14a] in ribonucleoprotein stability, and to the many excellent
contributions presented at the second annual Biophysics Conference, will help
establish the basic rules for fractionation of subcellular particles in a reproducible
manner.

The dependence of nucleoprotein structure upon divalent cation concentra-
tion is striking enough for these ions to become of interest in consideration of
the variables which dictate when a nucleic acid will be in the double helix,

GILLCHRIEST AND BOCK 9

when it will split, when it is "soluble RNA," and when not. Re-evaluation of
Brachet's [26] findings on the relation of ribonucleoprotein to growth phase
of yeast will be warranted in the light of current concepts of the importance
of buffer media.

Whereas the divalent cations of the buffer medium play an important role
in nucleoprotein structure, there appear to be mineral elements which are in-
fluential in nucleic acid function and are not in free equilibrium with the
buffer. Zittle [27] and Jungner [28] demonstrated that carefully isolated yeast
RNA contained characteristic amounts of metal ions. Kihlman [29] and
Mazia [25] have related structural integrity of chromosomes to metal content.
Loring and Cooper [30] find that certain cations are important for nucleopro-
tein stability of tobacco mosaic virus and hence for infectivity. Racker and
Krimsky [31] have evidence that metal ions are involved in an animal virus.
These are but a few of the many nucleoprotein-metal systems cited in the
literature.

Thus it appears that for viruses, as well as for subcellular particles, elucida-
tion of the structural and functional role of cations will be a fertile and chal-
lenging frontier for those with pioneering instincts.

SUMMARY

Ribonucleoprotein particles of sedimentation coefficient S2oo = S6 have been
isolated from A. vinelandii. The particles are free of lipid and DNA. They
are stable at neutral pH, in low-ionic-strength solution when divalent cations
are present; they are unstable in sucrose, in concentrated salts, and in the
presence of ribonuclease. Nucleic acid derived from the particles contains an
unidentified fifth base. The 86 S unit reversibly dissociates to particles of 58
and 39 S when the Mg++ concentration is lowered.

ACKNOWLEDGMENTS

We are pleased to acknowledge the generous aid of our colleagues. Profes-
sor Paul Kaesberg conducted the electron-microscope studies, Miss Vatsala
Thakur provided the N-terminal amino acid analyses, and Miss Fay Hoh col-
laborated in studies on the protein derived from the particles. Financial support
from the National Institutes of Health and Wisconsin Alumni Research Foun-
dation is gratefully acknowledged.

REFERENCES

1. L. Lilienfeld, Z. physiol. Chem., 18, Ab. A. Claude, Advances in Protein
473 (1894). Chem., 5, 423 (1949).

2. W. Huiskamp, Z. physiol. Chem., 32, 5. W. C. Schneider and G. H. Hoge-
145 (1901). boom, /. Biol. Chem. 183, 123 (1950).

3. R. R. Bensley and N. L. Hoerr, Anat. 6. N. G. Anderson, Science, 121, 775
Record, 60, 251 (1934). (1955).

4a. A. Claude, Harvey Lectures, 43, 121 7. P. Siekevitz and M. L. Watson, /. Bio-

(1948). phys. Biochem. CytoL, 2, no. 6, 653 (1956).

MICROSOMAL PARTICLES

8a. G. E. Palade and K. R. Porter, /. Ex-
ptl Med., 100, 641 (1954).

8b. G. E. Palade, /. Biophys. Biochem.
Cytol., 2, 547 (1954).

8c. G. E. Palade and K. L. Porter, /. Bio-
phys. Biochem. Cytol, 3, 269 (1957).

8^. G. E. Palade, /. Biophys. Biochem.
Cytol, 2, no. 4, Suppl., 85 (1956).

8<?. G. E. Palade and P. Siekevitz, /. Bio-
phys. Biochem. Cytol, 2, 171 (1956).

9. P. C. Zamecnik, Set. American, 5,
118 (1958).

10. D. P. Burma and R. H. Burris,
/. Biol. Chem., 225, 287 (1957).

11. A. J. Hodge, E. M. Martin, and
R. K. Morton, /. Biophys. Biochem. Cytol,
3, no. 1, 61 (1957).

12. Irving B. Sacks, personal communi-
cation.

13. J. Pochon, Y. T. Tchan, and T. L.
Wang, Ann. inst. Pasteur, 74, 182 (1948).

\4a. Fu-Chuan Chao and H. K. Schach-
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(1956).

\4b. Fu-Chuan Chao, Arch. Biochem.
Biophys., 70, 426 (1957).

15. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochem. Biophys., 38,
245 (1952).

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17. E. R. M. Kay and A. L. Dounce,
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Chemical Basis of Heredity (W. D. Mc-
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1957.

19. M. G. Sevag, D. B. Lackman, and
J. Smolena, /. Biol. Chem., 124, 425
(1938).

20. H. Fraenkel-Conrat, Virology, 4, 1-4
(1957).

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chim. ct Biophys. Acta, 20, 285 (1956).

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Gunsales, and S. Ochoa, /. Biol. Chem.,
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The Stabilization and Physical Characteristics of
Purified Bacterial Ribonucleoprotein Particles

JACK WAGMAN WESTON R. TRAWICK

U. S. Army Chemical Corps, Fort Detric\, Frederick., Maryland

Principally through electron-microscope studies on thin sections of various
species [1] it has been possible to show that a major part of the bacterial cyto-
plasm consists of widely dispersed granules 100 to 200 A in diameter. In ultra-
centrifugal analysis of aqueous extracts from disrupted bacteria, Schachman,
Pardee, and Stanier [2] found one of the major components, with j2o,w = 40 S,
to consist of roughly spherical particles, about 150 A in diameter, which con-
tain much of the cytoplasmic ribonucleic acid (RNA). A variety of enzymatic
functions have since been attributed to these particles [3], including the systems
for electron transport, for oxidative phosphorylation, and for some of the re-
actions of the tricarboxylic acid cycle.

The present paper reports attempts to isolate the 40 S component, by suc-
cessive differential centrifugation of extracts from Escherichia coli, and physi-
cal measurements obtained with purified material. Particular attention is drawn
to the observation that the stability of these particles is dependent upon a
dialyzable substance in cell extracts which apparently inhibits decomposition.

MATERIALS AND METHODS

The extracts used were prepared from E. coli (ATCC 4157) grown 24 hours
on nutrient agar at 37° C. After washing by alternate centrifugation and re-
suspension, cells were disrupted by shaking with glass beads in a Mickle dis-
integrator. The procedure consisted of shaking 6-ml volumes of suspensions
(about 7xl010 cells/ml) with 5 g of beads (type 114 Minnesota Mining and
Manufacturing Company) for 5-minute periods at 1700 cycles per minute. The
extracts were then cleared of unbroken cells and debris by low-speed centrifu-

MICROSOMAL PARTICLES

gation. Potassium phosphate buffer, pH 7.0 and ionic strength 0.1, was used in
the preparation of extracts.

Preparative and analytical sedimentation were carried out in Spinco ultra-
centrifuges Models L and E, respectively. A diffusion constant measurement
was made by free diffusion in a Claesson cell [4]. Partial specific volume was
determined by density measurements in a Lipkin pycnometer [5].

RESULTS

Decomposition Inhibitor in E. coli Extracts. It has been shown that the ex-
tractive procedure described here yields solutions that are highly reproducible
as determined by sedimentation and electrophoretic behavior [6]. The sedi-
mentation diagram (see fig. 1) corresponds closely to those obtained previ-
ously [2] by other methods of cell disruption. The rapidly sedimenting 40 S
component is clearly resolvable, and it appears that a considerably purified prep-
aration should be obtainable simply by successive differential centrifugation.
In early fractionation attempts, pellets from solutions subjected to centrifugal
fields about 100,000^ for 90 minutes (in the no. 30 rotor of the Model L Spinco)
were found to be only partly resoluble, in agreement with the finding of
Schachman et al. [2]. Moreover, the soluble material, as shown in figure 1,
contained an unexpectedly large amount of more slowly sedimenting material
along with a disappointingly low quantity of 40 S component.

In a subsequent study to determine the effect of dialysis on the nature of
E. coli extracts, an observation was made that proved to be an important step
in this purification problem. As shown in figure 2, the 40 S component in
dialyzed extracts was greatly reduced in concentration with a simultaneous in-
crease in the quantity of more slowly sedimenting material. In additional ex-
periments, however, the effect was found to be diminished as the dialysate-
extract volume ratio was decreased. This suggested the presence, in those
extracts, of a dialyzable material that functions as a stabilizer of the 40 S com-
ponent. A substance with an analogous property has been reported by Peter-
mann and Hamilton [7] in studies with rat liver homogenates.

Extract

Pellet fraction

Fig. 1. Sedimentation diagrams of an E.
coli extract and the pellet fraction derived
by a two-step centrifugation at 100,000^ for
90 minutes. Patterns were recorded 7 min-
utes after attainment of full field strength,
about 250,00%.

Extract

Extract dialyzed
against buffer

Fig. 2. The effect of dialysis on the sedi-
mentation behavior of an extract from E.
coli. Recording of diagrams took place 8
minutes after full field strength was reached,
250,000^.

WAGMAN AND TRAWICK 13

The stabilizing effect of the dialyzable material, which is tentatively desig-
nated "decomposition inhibitor" (DI), was further demonstrated as follows.
After overnight dialysis of 1 volume of extract solution against 4 volumes of
water, the dialysate was lyophilized and reconstituted to four-fifths of the vol-
ume of extract used. This procedure yields a solution, which we shall refer
to as buffer-DI, whose concentration of dialyzable material is roughly equal to
that of the original extract. The stabilizing effect of the DI was then observed
by comparing the sedimentation behavior of fresh extract solutions dialyzed
against buffer and buffer-DI, respectively. As is shown in figure 3, the DI
effectively prevented the disappearance of 40 S component.

The nature of the stabilizing substance is not clear. An analysis of the di-
alyzable material indicates the presence of peptides and nucleotides as well as
trace amounts of magnesium, iron, and other metals usually found in bacterial
extracts. Metal ions appear to be ruled out as the active substance, for the sta-
bilizing effect is lost by heating the buffer-DI for 5 minutes at about 90° C.

Purification of 40 S Component. It now appears that the early difficulties in
fractionating the 40 S component were due to a procedure which separates that
component from the stabilizer. By slightly modifying the original fractionation
scheme, the 40 S component was prepared in a relatively high state of purity
and stability. The pellets, after each of two successive centrifugation steps, were
redissolved in previously prepared buffer-DI. Figure 4 shows a comparison of
preparations, from a single batch of cells, obtained by this and the previous
methods. The sedimentation patterns demonstrate the degree of purity with
which it is possible to obtain the 40 S component, and illustrate the activity of
the DI.

Physicochemical Properties. An analysis of the fractionated material indicated

Extract

Extract dialyzed against:
Buffer Buffer-DI

Fig. 3. Sedimentation diagrams illustrating the protective effect of the dialyzable stabi-
lizer on the 40 S component in E. coli extracts.

MICROSOMAL PARTICLES

Crude extract

Centrifugal! y fractionated RNP (40 S) component

Without buffer-DI

Using buffer-DI
observed initially

Using buffer-DI
observed 4 days later

Fig. 4. Sedimentation diagrams illustrating the influence of the stabilizing medium
(buffer-DI) on the nature of the RNP fraction obtainable from E. coli extracts by differen-
tial centrifugation. Diagrams were recorded about 5 minutes after full field strength was
reached, 250,00%.

that it consisted entirely of protein and ribose nucleic acid in a proportion about
3 to 1 by weight. Since the material appears to be essentially monodisperse, it
is tentatively concluded that the 40 S particles contain protein and nucleic acid
in combination as a ribonucleoprotein (RNP) .

Sedimentation, diffusion, and partial specific volume measurements were
carried out with the RNP fraction, the more detailed data being obtained with
water as the solvent. The results are summarized in table 1. Although, as is
seen in figure 4, very small amounts of more slowly sedimenting material were
present in these preparations, they are believed not to cause serious errors in
the use of these data to determine particle characteristics.

TABLE 1. Sedimentation and Diffusion Constants of Centrifugally Fractionated
Ribonucleoprotein from Extracts of E. coli

Concentra-
Prepa- tration

ration Solvent mg/ml

a 0.1 ionic strength 6.3
phosphate buf-
fer, pH 7.0
b Water 8.0

6.0
2.0
0

11)

13,

•*20, W

sec

45.0

43.6
44.4
46.0

46.9 *

107D2(V

cm- sec

-l

2.67

V20

0.657

1,240,000

f/fo

.16

* Value obtained by extrapolating \/s.

WAGMAN AND TRAWICK 15

The very slight concentration dependence of S20, w (with a value, at infinite
dilution, of 46.9 S) is in agreement with the finding by Schachman et al. [2] that
these particles appear to be roughly spherical in the electron microscope. On
the assumption of a negligible concentration dependence for diffusion, the
molecular weight calculated from the measured data is 1,240,000, and the fric-
tional ratio, f/fo, is 1.16, corresponding to spheres, 137 A in diameter, hydrated
to the extent of 0.37 ml/g of RNP.

An electron micrograph, made using a water solution of RNP, is shown in
figure 5. The particles appear to be uniformly spherical with a mean diameter
about 152 A. This value is in good agreement with the sedimentation-diffusion
figure, inasmuch as the probable errors with the electron microscope tend to
yield too high a figure.

DISCUSSION

It has been reported (e.g., by Chao and Schachman [8] and by Bolton, Hoyer,
and Ritter, paper 3 of this volume) that more rapidly sedimenting components
(60 S and 80 S) appear in extracts from microbial cells when magnesium ions
are present in sufficient concentration in the extract media. This addition to
the ionic environment is not required for the preservation of the 47 S particles
dealt with in the present report. In the intact cell, the synthesis and dissocia-
tion of RNP particles are probably controlled by several factors which may
include magnesium and other ions. The action of ribonuclease, for example,
is inhibited by a large number of substances [9] including bivalent cations and
mononucleotides, all of which are apparently present in the dialyzable fraction
of E. coli extracts.

Although the RNP component has been identified with a variety of enzymatic
functions, it is not clear whether these are intrinsic or merely adsorbed. Elec-
trophoretic data [6] show that this material, despite its apparent monodispersity
in the ultracentrifuge and electron microscope, is heterogeneous, consisting of
two or more components with a wide range of anodic mobilities at pW 7.0.

ACKNOWLEDGMENT

We wish to acknowledge the assistance of Mrs. F. Elizabeth White in the
growth and harvesting of the E. coli cultures and of Mr. Robert L. Sine in the
preparation of the electron micrograph.

SUMMARY

A dialyzable substance in extracts from E. coli has been found to inhibit
the decomposition of the RNP particles (previously referred to as the 40 S
component). This observation has led to the preparation of the RNP fraction
in a relatively high state of purity and stability. Physical measurements indi-
cate that the RNP component consists of uniformly spherical particles with
a molecular weight 1,240,000 and a diameter of 137 A.

MICROSOMAL PARTICLES

Fig. 5. Electron micrograph taken with a specimen of purified RNP. Shadow angle
3:1, RCA electron microscope. Polystyrene latex particles, 0.188-micron diameter, were
used as internal standard.

WAGMAN AND TRAWICK

REFERENCES

1. J. R. G. Bradfield, "Organization of
bacterial cytoplasm," from Bacterial Anat-
omy, Cambridge University Press, 1956.

2. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochem. Biophys., 38,
245-260 (1952).

3. M. Alexander, Bacteriol. Revs., 20,
67-93 (1956).

4. S. Claesson, Nature, 158, 834 (1946).

5. Ace Glass Company, Vineland, New
Jersey.

6. J. Wagman, E. Pollack, and E. J.
Weneck, Arch. Biochem. Biophys., 73, 161-
170 (1958).

7. M. L. Petermann and M. G. Hamil-
ton, /. Biophys. Biochem. CytoL, 1, 469-
472 (1955).

8. F.-C. Chao and H. K. Schachman,
Arch. Biochem. Biophys., 61, 220 (1956).

9. M. R. McDonald, in Methods in En-
zymology (Colowick and Kaplan, eds.),
vol. 2, pp. 433-434, Academic Press, New
York, 1955.

Stability of Ribonucleoprotein Particles

of Escherichia coli

ELLIS T. BOLTON

Department of Terrestrial Magnetism-
Carnegie Institution of Washington

BILL H. HOYER DANIEL B. RITTER

Rocky Mountain Laboratory

National Institute of Allergy and Infectious Diseases

U. S. Public Health Service, Hamilton, Montana

Investigations concerned with the structure and function of ribonucleopro-
teins of microorganisms require particle preparations that are representative,
reproducible, and stable. This report presents some results of exploratory
studies in which the analytical ultracentrifuge was used to assess the influence
of various suspending media on the ribonucleoproteins of "Escherichia coli.

METHODS

E. coli, B (ATCC 11303) harvested during the exponential phase of growth
in a glucose-salts culture medium * was used for all studies. The bacteria were
washed and resuspended (25 mg dry weight of bacteria per milliliter) in ap-
propriate buffer solutions and disrupted by means of a modified French pres-

1 The composition of C medium and other culture conditions may be found in
Roberts et al., Studies of Biosynthesis in Escherichia coli, Carnegie Inst. Wash. Publ. 607,
Washington, D. C, 1955.

BOLTON, HOYER, AND RITTER

sure cell 2 operated at approximately 10,000 psi. Break-
age of the bacteria by this means is essentially com-
plete. The resulting bacterial juices were examined in
the analytical ultracentrifuge (Spinco, Model E) as
soon as practicable (about 30 minutes after rupture),
or after various periods of storage at 4° C. The
centrifuge was routinely brought up to speed in 6 to
7 minutes and held at about 60,000 rpm for the dura-
tion of the run.

RESULTS

Figures 1 to 4 are illustrative sedimentation dia-
grams showing that the pattern of rapidly sediment-
ing components varies in accord with the kind of
suspending medium used. Figure 1 compares the
sedimentation behavior of the components in extracts
prepared from bacteria broken in 0.01 M Tris-0.004 M
succinic acid-0.005 M magnesium acetate buffer (pH.
7.6, "TSM"), in 0.01 M Tris-0.004 M succinic acid
("TS"), or in TSM + 0.07 M phosphate (pH 7.6).
Values spotted along the abscissa are approximate ap-
parent sedimentation coefficients. It is evident from
this comparison that more, and larger, components
are observed when magnesium has been included in
the buffer and also that phosphate abolishes the more
rapidly sedimenting materials. Whether the effect of
phosphate is specific or whether the result is due to an
increased ionic strength of the medium is not known.
The sharp spike characteristic of highly polymerized
deoxynucleic acid (DNA)3 is missing from these dia-
grams, although it is readily observed in juices pre-
pared by breaking E. coli as a result of lysozyme treat-
ment and osmotic shock. In spite of this finding,
three-quarters of the ultraviolet-absorbing substance
and one-seventh of the protein of E. coli disrupted in
the TSM medium may be sedimented in the prepara-
tive rotor (100,000g, 90 minutes).

Figure 2 shows that juices prepared by pressure
cell disruption maintain constant sedimentation dia-
grams for at least 20 hours. If, however, sodium
ethylenediaminetetraacetate (EDTA, 0.1 M, pH. 7.6) is
extracts, all components greater than about 20 S disappea

TSM +
07M PO,

iltaW

8 mm 60,000 RPM

Fig. 1. Sedimentation
diagrams of E. coli dis-
rupted in various buffer
solutions. The concentra-
tion of the bacterial juices
differed among the runs.

added to the bacterial
r. This occurs whether

2 C. S. French and H. W. Milner, Methods in Enzymology I, Academic Press, p. 65. A
similar device is marketed by the American Instrument Company, Silver Spring, Maryland.

3 See, for example, the sedimentation diagrams reported by W. Gillchriest and R. Bock,
S. Dagley and J. Sykes, and J. Wagman reported in the present volume.

MICROSOMAL PARTICLES

0 hours

20 hours

12 min 60,000 RPM

Fig. 2. Influence of storage at 4° C. The suspending buffers (TSM, left; TS, right)
also contained 0.25 M sucrose, although subsequent runs have shown that sucrose has no
effect on the pattern of components.

or not magnesium is included in the original suspending medium, as figure 3
demonstrates. In addition, a markedly decreased (<10 per cent) ultraviolet
absorption occurs in the 100,000^-1 hour pellets (preparative rotor) when
EDTA has been added to the bacterial juices. Figure 4 shows that DNAase
(2 ug/ml) has little, if any, effect upon the number and size of the rapidly
sedimenting materials, whereas RNAase (approximately 10 ug/ml) removes
these components.

SUMMARY AND CONCLUSIONS

Pressure cell disruption of E. coli at pH 7.6 in magnesium-containing solu-
tions of low ionic strength (e.g., 0.01 M Tris-succinate) releases high-molecu-
lar-weight components which range from 20 to 80 S. These components "fall
apart," i.e., become elements having sedimentation coefficients less than about
20 S, when a chelater, EDTA, or the enzyme ribonuclease is allowed to act
upon them. DNAase, sucrose, or cysteine exerts no apparent effect on either
the number of components or their relative quantities. Nearly all (>80 per
cent) of the ribonucleic acid and about one-seventh of the protein of E. coli
can be sedimented in a preparative rotor under optimum conditions (TSM,
100,000g, 90 minutes). No RNA and only a trivial amount of protein can be
sedimented after EDTA or ribonuclease treatment. Hence, it may be con-

BOLTON, HOYER, AND RITTER

EDTA

JJLJ

6 mm 60,000 RPM

Fig. 3. Effect of EDTA on the sedi-
mentation diagrams of E. coli juice. The
two lower diagrams are from preparations
containing one-half as much material as
those for the upper pattern.

6 min 60,000 RPM

Fig. 4. Effect of nucleases on sedimenta-
tion diagrams. The lower pattern is from a,
preparation one-half as concentrated as that
of the upper diagram.

eluded that the bulk of the high-molecular-weight components of E. coli is
composed of ribonucleoproteins held together in a fashion in which divalent
cation (s) (probably Mg++) and the integrity of ribonucleic acid play important
roles. Thus, in certain physical and chemical attributes the "ribosomes" (ribo-
nucleoprotein particles) of E. coli resemble constitutive elements of the cyto-
plasm of other bacteria, and also of yeast, plants, and mammals.

Biochemical Characterization and

Electron-Microscopic Appearance of

Microsome Fractions

DAVID GARFINKEL1

Eldridge Reeves Johnson Foundation for Medical Physics
University of Pennsylvania

The electron-microscope studies on microsomes by Palade and Siekevitz [1]
have resulted in the definition of three kinds of microsomes: granules of 150 A
diameter; smooth-surfaced vesicles; and rough-surfaced vesicles which differ in
appearance from the smooth-surfaced ones primarily by having the granules
attached to them. Biochemical studies of microsomes have resulted in the iso-
lation of two varieties of microsomes — the 150 A granules just mentioned,
which are the principal subject of interest in this symposium, and which are
rich in RNA but poor in lipid and cytochrome b5, and a particle isolated by
Perm and Mackler [2] which is rich in cytochrome b5 and lipid and poor in
RNA. It will be shown here that there are at least three biochemically dis-
tinct varieties of microsomes, correlated with those observed in the electron
microscope.

It is possible to fractionate mammalian liver microsomes (the work here
described is with pig liver) so as to obtain, in addition to the microsomes as
they are usually prepared, a small light fraction of microsomes which is usually
found to contain about twice as much cytochrome b5 and less than half as
many ribonucleoprotein granules per unit biuret protein as the bulk of the
microsomes, hereafter referred to as the bul\ fraction. This fractionation may
be made, for instance, by centrifuging a concentrated (1 part liver to 2 parts
0.25 M sucrose) homogenate, after the mitochondria have been removed, at

1 Public Health Service Research Fellow of the National Cancer Institute, 1955-1957.

GARFINKEL 23

70,000^ for 25 minutes. In addition to the bulk microsomal pellet, the light
fraction is obtained as a suspension at the bottom of the centrifuge tube. In
order to permit spectroscopic study, both fractions are washed with Ringer's
solution, which removes the hemoglobin. It also washes out about 80 per cent
of the RNA (although the ribonucleoprotein granules do not disintegrate and
may still be isolated [3]), so that there are no accurate values for RNA con-
centration. It is found, however, that the bulk fraction contains appreciable non-
cytochrome heme, somewhat more than the cytochrome heme, whereas the light
fraction contains very little. A detailed description will be published elsewhere.

We have seen that the microsomes that centrifuge down last, the light frac-
tion, are rich in cytochrome b5. Palade and Siekevitz [1] found that they were
likely to be smooth-surfaced. This finding suggests a correlation between
smooth-surfaced appearance and the presence of cytochrome b5. So does the
fact that studies [4] in various tissues indicate that much of the cytochrome
is present only where the electron microscope shows smooth-surfaced endo-
plasmic reticulum.

Drs. Ian R. Gibbons and T. F. Anderson kindly took photographs of one
of these microsomal preparations with the electron microscope. Figure 1 shows
views of the light and bulk fractions. It is seen that there is much more ma-
terial of smooth-surfaced origin (free of the ribonucleoprotein granules) in the
light fraction than in the bulk fraction. Counts of the numbers of smooth- and
rough-surfaced microsomes, using unfixed preparations to avoid any enrich-
ment of one microsomal type in the process of fixation, indicated that the per-
centage of smooth-surfaced microsomes was proportional to the cytochrome con-
centration (a light fraction which was twice as rich in cytochrome as the bulk
fraction contained twice as many smooth-surfaced microsomes). Apparently
cytochrome b5 is localized in the smooth-surfaced microsomes and the rough-
surfaced ones contain little of it. This observation is also in agreement with
the fact that predominantly rough-surfaced microsomes can be prepared (from
pancreas, for instance [4, 5]), and they contain little or no cytochrome. It
would be desirable to confirm this by preparing pure smooth- or rough-surfaced
microsomes and finding their cytochrome content (since the preparative method
used enriches the smooth-surfaced microsomes only twofold, the resulting con-
clusions regarding their properties should not be considered final).

We are not limited to the electron microscope, but can also study the micro-
somes by biochemical methods. The procedure used here is digestion with
pancreatin, a mixture of digestive enzymes from the pancreas. After digestion,
which is not complete, what is left of the microsomes is centrifuged down and
examined.

Initially, two suspensions of light and bulk microsomes have equal con-
centrations of cytochrome. Assuming that the smooth-surfaced microsomes
contain nearly all the cytochrome, then their concentration is equal in the two
suspensions. The protein concentrations are shown in table 1. The digestion

Fig. 1. Electron micrographs of light (above) and bulk (below) fraction microsomes,
fixed with osmium tetroxide and air-dried from distilled water. Magnification 20,000 X .
The rough-surfaced microsomes may be identified by the little particles attached to them.

GARFINKEL

TABLE 1

Sedimentable Protein, mg/ml

Before digestion
After digestion

Light Fraction

Bulk Fraction

35
20

for 10 days to 2 weeks is sufficient to solubilize nearly all the cytochrome;
this is in fact the method of preparing cytochrome b5 [6, 7]. The nucleopro-
tein granules have practically disappeared in the process. Examination of the
sedimentable protein content of these two suspensions shows that much more
protein is left in the bulk fraction. Nearly all the nucleoprotein has been re-
moved, and since the cytochrome contents were equal there should be the same
amount of residue from the smooth-surfaced microsomes in both fractions.
Apparently there is something present in the bulk fraction (of which there is
much less in the light fraction) to account for the difference in protein con-
tent. Since the other two forms were eliminated, this must be the rough-sur-
faced microsomes. That there is a qualitative difference in the residues can be
seen by looking at the centrifuged pellets. Both contain a transparent amber
layer, but this is all that is left of the light microsomes, whereas the bulk-frac-
tion pellet has below this transparent layer an opaque tan one, showing some
signs of further layering. This bulk-fraction pellet still contains the noncyto-
chrome heme originally present. The rough-surfaced microsomes, which have
not previously been characterized biochemically, are therefore found to contain
a tan pigment and the noncytochrome heme, and to be distinct from the smooth-
surfaced ones. This finding is summarized in table 2. It should be kept in mind
that the expression "rough-surfaced microsomes" means the vesicles themselves

TABLE 2

Cross-sectional

Appearance
in Electron
Microscope

Isolated
by

This Pai

-tide Is

Particle

Rich in

Poor in

Ribonucleo-
protein
granule

Small,
filled-in
circle

Petermann
and
Hamilton [3]

RNA

Cytochrome b5,
noncytochrome
heme

Smooth-
surfaced
microsome

Empty
circle
or
ellipse

Penn and
Mackler [2]
(probably a
fragment)

Cytochrome b5,
lipid

Noncytochrome
heme
(RNA?)

Rough-
surfaced
microsome

Empty circle
or ellipse
with small
filled-in cir-
cles attached
to outside

Lipid,
noncytochrome
heme,
tan pigment

MICROSOMAL PARTICLES

without the attached granules. The fact that three varieties of microsomes have
been defined is not intended to imply that any of these varieties of microsomes
is itself homogeneous.

REFERENCES

1. G. E. Palade and P. Siekevitz, /. Bio-
phys. Biochem. CytoL, 2, 171 (1956).

2. N. Penn and B. Mackler, Federation
Proc., 16, 232 (1957).

3. M. L. Petermann and M. G. Hamil-
ton, /. Biol. Chem., 224, 725 (1957).

4. D. Garfinkel, unpublished experi-
ments.

5. G. E. Palade and P. Siekevitz, /. Bio-
p/iys. Biochem. CytoL, 2, 671 (1956).

6. D. Garfinkel, Arch. Biochem. Bio-
phys., 70, 111 (1957).

7. P. Strittmatter and S. F. Velick,
/. Biol. Chem., 221, 253 (1956).

The Configurational Properties of

Ribonucleic Acid Isolated from
Microsomal Particles of Calf Liver

BENJAMIN D. HALL PAUL DOTY

Department of Chemistry, Harvard University

Although ribonucleic acids (RNA) from many sources have been examined
by physical methods within the last few years no clear and consistent picture
of the configurational properties of RNA has materialized. Most studies of
RNA have been complicated by spontaneous changes of molecular weight, ag-
gregation under some conditions and degradation under others. In the work
reported here we have avoided these complications by finding experimental con-
ditions under which the RNA is stable and have then proceeded to establish
its configurational properties in solution by means of several different physical
methods.

The choice of microsomal particles from liver as our source of RNA was
prompted by the particular importance that these ribonucleoprotein particles
have assumed by virtue of their participation in protein synthesis [1, 2] and the
fact that they can be isolated in pure form [3] before the preparation of the
RNA itself.

It is important to emphasize at the outset that our major emphasis in the
work reported here has been on the configurational properties of stable RNA
isolated from these particles. We defer until a later time a report on the molecu-
lar weight and configuration of RNA within the microsomal particles and the
relation of the work presented here to these properties.

THE PREPARATION OF RNA

Preparation of Microsomal Particles. The procedure summarized below
evolved from those used by Zamecnik et al. [1] and by Petermann and Hamil-
ton [4] for the isolation of similar particles from rat liver. Calf liver was

MICROSOMAL PARTICLES

quickly frozen in Dry Ice within 3 minutes of slaughter, and thawed immedi-
ately before proceeding with the preparation. The liver was thoroughly chopped
while thawing and the cell walls were broken by blending in an Osterizer
Blendor intermittently for 3 to 4 minutes at about half speed. The suspending
medium for this operation was ice-cold 0.25 M sucrose (2 cc/g liver).

The resulting suspension was centrifuged twice for 30 minutes at 1500^ in
the cold. The supernatant solution, containing the microsomes, glycogen, and
soluble liver proteins, was removed by pipet after each centrifugation. The
microsomal particles were then separated from the lipoprotein portion of the
microsomes by emulsifying the microsomes with sodium deoxycholate [1]. A
pellet of microsomal particles can then be obtained by ultracentrifugation of
the deoxycholate-treated microsome suspension. To the supernate from the
second low-speed centrifugation, 1/9 volume 5 per cent sodium deoxycholate
(in 0.05 M Tris buffer, pH 8.2) was added with stirring. Stirring was con-
tinued for 15 minutes at 0° C. The microsomal particles were sedimented by
centrifugation for 5 hours at 29,000 rpm in the no. 30 rotor of a Spinco model L
ultracentrifuge. The dark red supernatant solution was removed from the
microsomal-particle pellet by decantation. The pellet was used without further
purification for preparing RNA. For studies on the microsomal particles, fur-
ther centrifugation, both high- and low-speed, was employed to obtain micro-
somal particles free from contaminating proteins.

Properties of the Microsomal Particles. The degree of homogeneity of the
particles is revealed by the sedimentation diagram shown in figure 1. Extra-
polation of numerous measurements at various concentrations in the 0.025 M

Fig. 1. Sedimentation diagram of microsomal particles in 0.025 M NaHCOs, 0.004 M
MgCl2, pli 8.5. Picture taken at bar angle of 32° after 24 minutes at 27,690 rpm. ^20 = 78.7
at this concentration (1.2 g/dl) ; ^°20 = 81.3.

HALL AND DOTY 29

NaHC03 containing 4 mM MgCl2 (pH 8.5) yielded 81 for i°2o,w. The ratio of
optical densities at 260 and 280 mp was 1.80 in this solvent. The ratio at 260
and 230 mu was 1.32. Various mild treatments such as heating to 37° C cause
the appearance of 50 S and 5 S components accompanied by increases in the
optical density at 260 mp (resembling the denaturation of DNA) and conse-
quently increases in the values of the optical density ratios reported above.

Preparation of RNA from the Microsomal Particles. In order to obtain RNA
of high purity from microsomal particles, two principal steps must of neces-
sity be included in the procedure: (1) dissociation of the RNA from the pro-
tein; (2) separation of denatured proteins and other contaminating substances
from the nucleic acid. This procedure makes use of one anionic detergent,
sodium lauryl sulfate, to disrupt the protein-nucleic acid complex (by denatur-
ing the protein and displacing the nucleic acid from cationic groups on the
protein), and another, sodium xylene sulfonate (Naxonate), to remove the
denatured protein from solution [5].

The pellet of microsomal particles was suspended in 0.01 M versene, pH 7.0.
In a typical preparation, beginning with 200 g of liver, the volume of the sus-
pension was 100 ml. The suspension was brought to 20° C, and sufficient solid
sodium lauryl sulfate was added (with stirring) to bring the concentration to
4 per cent. Stirring was continued until a clear solution resulted; this was
allowed to stand for 12 hours at 20° C. At the end of this time, the solution
was cooled to 5° C, and to it were added 3 volumes of an ice-cold solution of
0.2 M KCl, 0.01 M versene pll 7.0 containing 12 g sodium xylene sulfonate per
100 cc. The pll of this mixture was reduced to 4.3 by dropwise addition of 6 TV
acetic acid. After standing 15 minutes at 0°, the suspension was centrifuged
for 30 minutes at 1500g- in the cold. The supernatant solution was decanted
and brought to pH 7.0 by addition of 6 N NH4OH. After the solution was
warmed to 20° C, RNA was precipitated with 2 volumes isopropyl alcohol.
The precipitate was allowed to settle for 2 hours; then it was centrifuged down.
The liquid was decanted, and the RNA was dissolved in 0.03 M sodium acetate
solution. This solution was treated with Naxonate to complete the removal of
protein. To it were added 3 volumes 40 per cent Naxonate; then the solution
was stirred for 30 minutes. After cooling to 0° C, the pH was brought to 4.3
and the solution was filtered through celite and sintered glass.1 This treatment
removed the protein-Naxonate complex, which is insoluble, leaving glycogen
as the only nondialyzable impurity in the RNA. Glycogen may conveniently
be removed by centrifugation for 20 minutes at 30,000^ (it forms a pellet).
After the removal of glycogen, the RNA solution was dialyzed against 0.01 M
KH2PO4-K2HPO4 (1:1) in order to remove ultraviolet-absorbing impurities.

1 It has recently been found that a substantial part of the RNA (up to 85 per cent of the
total) is lost in this step because of adsorption on the celite. Besides lowering the yield,
this may have led to fractionation of the RNA, if the adsorption was selective. This step
may be omitted, for the protein-Naxonate aggregates can be removed along with glycogen
in the centrifugation at 30,000^.

MICROSOMAL PARTICLES

THE STABILIZATION OF RNA

The RNA prepared in the manner described above exhibited values of sedi-
mentation constant and intrinsic viscosity that depended very much on the
solvent employed, and invariably these quantities slowly diminished with time.
Choosing 0.01 M phosphate buffer at pYL 7.0 as solvent, typical preparations
would have initially sedimentation constants of 9 and intrinsic viscosities of 0.6.
The sedimentation pattern as observed in ultraviolet optics was rather broad
and usually single peaked. The use of the above values in the Mandelkern-
Flory equation [6] gave approximately 200,000 as the molecular weight.

In studying the gradual decay in molecular weight it was observed that heat-
ing to 60° C for a few minutes or the addition of KCNS to the extent of 0.2 M
would cause a 30 per cent fall in molecular weight. This indicates that the
polynucleotide chains of the original material were aggregated to a small but
significant extent.

When the RNA was heated to progressively higher temperatures in the
0.01 M phosphate buffer the molecular weight was observed to continue falling
up to temperatures of about 80° C. After exposure to 80° the molecular weight
remained unchanged with time at room temperature, for prolonged periods at
83° and for short periods at 95°. Thus a single exposure to about 80° C pro-
duced a stabilization of the RNA.

The changes which such heating induced in the sedimentation pattern are
shown in figure 2, where the sedimenting boundaries observed with ultraviolet
optics under identical conditions are shown for 0, 5, 15, and 45 minutes' heating
at 83° C. It is seen that the effect of the heating is to lower and to narrow the

I oo

0 75

C/c0
0 50

0.25

000

6.0

CHANGES IN SEDIMENTATION BOUNDARY
PRODUCED BY HEATING RNA TO 83°C

Relotive concentration versus
distance from center of rototion

Solvent -OIM KH2P04- K2HP04 , pH 7
RNA concentration 38 if/cc.

6.2 6 4

DISTANCE I N CM.

6 6

Fig. 2. Effect of heating upon RNA sedimentation boundaries after 21 minutes at
59,780 rpm.

HALL AND DOTY 31

sedimentation distribution; the change is nearly completed during the first 5
minutes of heating. The intrinsic viscosity showed a similar behavior. Both
these changes indicate a fall in apparent molecular weight to a limiting value.
The sharpening of the sedimentation distribution suggests a sharpening of the
molecular-weight distribution as well. Consequently the fall in molecular
weight appears to result from the dissociation of an aggregate and not the hy-
drolysis of phosphate ester bonds.

In order to proceed with the investigation of RNA stabilized by heating, a
standard procedure was adopted: solutions in 0.01 M phosphate buffer were
heated at 83° C for 10 minutes and then cooled to room temperature for
measurement.

PROPERTIES OF RNA

The intrinsic viscosity and sedimentation constant measured for a number
of preparations are listed in table 1. Recent results, shown in the lower part
of the table, have consistently given sedimentation constants near 7 S, in con-
trast to variable and lower results obtained earlier, which are listed in the upper
part of the table. RNA preparations having these higher and more consistent
sedimentation values were obtained by improving the separation of the micro-
somal particles from other cellular fractions (thereby reducing ribonuclease
contamination) and by maintaining a temperature of 5° or less at all stages
of the preparation in which RNA might be attacked.

For a typical, improved preparation of stabilized RNA with s02j = 6.6 S and
the intrinsic viscosity [•/]] =0.29, a molecular weight (weight average) of 106,000
can be calculated from the Mandelkern-Flory equation [6] using 2.35 X 106 for
(4>1/3/P) and 0.55 for the partial specific volume. Light-scattering measure-
ments on the same sample yielded the same value for the weight-average
molecular weight.

TABLE 1. Sedimentation Constant, Intrinsic Viscosity, and Molecular
Weight of RNA Preparations

Measured in 0.01 M phosphate, pH 7.0, after heating to 83° for 10 minutes.

*°25 M25 Ms3w

2.9 0.14 21,700

4.1 0.17 40,700
4.6 0.19 50,400

5.0 ...

6.2 0.26 93,000
6.6 0.29 106,000
6.6 ...

7.1 ...

7.4 0.27 120,000

6.8 ±0.4 0.27 106,000

MICROSOMAL PARTICLES

The agreement between the light-scattering molecular weight and that cal-
culated from sedimentation and viscosity data justifies the application of
the Mandelkern-Flory relation to the lower-molecular-weight samples listed in
the upper part of table 1. The molecular-weight dependence of these two quan-
tities can then be examined. This is done in figure 3, where the logarithms of
s° and [y\] are plotted against the logarithm molecular weight, yielding the
linear relations

/=2.1xl0"2 M0A9
[n]=:6.2xl0-4 M0-53

This type of dependence is associated with homologous samples of linear, ran-
domly coiled polymer chains. These exponents are close to the limiting value
of 0.5 which is reached for chains having the maximum permissible extent of
coiling [7], So high a degree of coiling is unexpected in a highly charged poly-
electrolyte at the relatively low ionic strength used here and must be taken to
indicate that the intrachain attractions are strong enough to overcome the ex-
pansive electrostatic effect.

Provided that RNA is a randomly coiled, single chain, we should expect the
relatively tight coiling to give way to a much more expanded coil in the ab-
sence of added electrolyte. This would be recognized by a much higher viscosity
and the further increase in the reduced specific viscosity upon dilution with
water, a behavior known as the electroviscous effect in polyelectrolyr.es. When
the RNA is transferred to aqueous solution (pH 5), its reduced specific
viscosity at 0.6 g/dl is found to be 0.85 and to increase strikingly upon dilution.
These results, shown in figure 4, clearly indicate the progressive expansion of the

10000

20,000

40,000

100,000

Fig. 3. Logarithmic dependence of the sedimentation constant and intrinsic viscosity of
RNA upon the molecular weight.

HALL AND DOTY

molecule as its counterions become further removed from it. Upon the addi-
tion of salt the viscosity returns to a low value, showing the reversibility of
the effect. Thus the solution properties of the RNA are found to be satisfac-
torily correlated with a randomly coiled, single-chain structure.

THE HYPOCHROMICITY OF RNA

In view of the substantial nucleotide attractions that appear to be necessary
to account for the relatively tight coiling of RNA in 0.01 M phosphate buffer
we sought to examine the possibility that hydrogen bonds between pairs of
nucleotides were present in a random fashion within each coiled RNA mole-
cule. Since the break-up of hydrogen bonds between base pairs in the denatura-
tion of deoxyribose nucleic acid is accompanied by a substantial rise in ultraviolet
absorbance [8], it seemed reasonable to look for the same effect in RNA. The
hydrogen bonds that may exist in RNA would be broken during the expansion
that was seen to accompany the removal of salt from the RNA solution. Alter-
natively such hydrogen bonds may be expected to be broken with increasing
temperatures, as occurs with deoxyribose nucleic acid. We have combined these
two hydrogen-bond-breaking effects by measuring the optical density of RNA
solutions at 258 mu as a function of temperature at various ionic strengths.

The results of this study are shown in figure 5. It is seen that in 0.01 M

675

.650-

.625

.600 -

.575

0.2 0 4 0 6

RNA CONCENTRATION, gms/lOOcc

Fig. 4. Reduced specific viscosity of
RNA in water; dependence on concen-
tration.

100

Fig. 5. Variation of the optical density of
RNA solutions at 258 m« with temperature and
ionic strength.

34 MICROSOMAL PARTICLES

phosphate buffer the optical density rises at once as the temperature is raised
above room temperature and reaches a maximum value, 28 per cent higher, at
about 80° C. At higher ionic strength the rise is similar but does not begin
until a higher temperature is reached. At lower ionic strength the optical
density at room temperature is already considerably above the lower limiting
value. Thus at the reduced ionic strength, where viscosity measurements show
that the molecule is partly expanded, these measurements indicate that the
hydrogen bonding is correspondingly reduced. These observations are consist-
ent with the initial hypothesis and support the view that the intrachain attrac-
tions in RNA arise from hydrogen bonding between the purine and pyrimidine
bases. The pairing would presumably be similar to that existing in DNA, but
this fact carries no implication that the base pairs would be periodically
organized.

Since the magnitude of the hypochromic effect (about 28 per cent) is more
than half the total that results from the hydrolysis of RNA [9], the implica-
tion is that a large fraction of the purine and pyrimidines participate in the
pairing when the ionic strength is 0.01 M or more. The fraction of base pairs
involved appears to be unchanged by the heating cycle. Indeed, the first heat-
ing cycle of freshly prepared RNA shows the same results as successive cycles,
in contrast with DNA, where the optical density never returns to the original
value after the first heating. One would therefore conclude that there is no
periodic arrangement of base pairs in RNA as there is in DNA.

Finally, it is of interest to note that the heat treatment used to stabilize the
RNA, heating to 83° C of a 0.01 M phosphate buffer solution, is precisely the
treatment required to reach the maximal optical density and presumably break
the hydrogen bonds. Consequently the drop in apparent molecular weight pro-
duced by the heat treatment may have done nothing other than permit some
aggregates of RNA molecules to be dissociated through the opening-up of the
hydrogen bonds holding them together. It remains for future work to show
whether or not this interchain bonding is a remnant of structural organization
of RNA in the microsomal particle.

ACKNOWLEDGMENT

We are deeply indebted to Dr. Norman S. Simmons, Atomic Energy Project,
University of California at Los Angeles, for his helpful advice and discussions
regarding the preparation of RNA. One of us wishes to thank also the Na-
tional Science Foundation and the Union Carbon and Carbide Corporation for
fellowship support during the course of this investigation. This investigation
was supported by the National Institutes of Health (C2170) .

Note Added in Proof

Using aqueous phenol for removal of protein, we have isolated from calf-liver
microsomal particles RNA of substantially higher molecular weight (~1 X 10°)
than that obtained in detergent RNA preparations. This large RNA appears

HALL AND DOTY

to be composed of subunits which are single-chain coils of the type described
here. When it is heated to 85° in 0.02 M phosphate buffer, the molecular weight
falls to 120,000, the intrinsic viscosity to 0.22, and the sedimentation constant
to 7.5.

REFERENCES

1. J. W. Littlefield, E. B. Keller, J. Gross,
and P. C. Zamecnik, /. Biol. Chem., 211 ,
111 (1955).

2. P. C. Zamecnik, E. B. Keller, M. B.
Hoagland, J. W. Littlefield, and R. B.
Loftfield, Ciba Foundation Symposium on
Ionizing Radiations and Cell Metabolism,
pp. 161-168, Churchill, London, 1956.

3. M. L. Petermann, N. A. Mizen, and
M. G. Hamilton, Cancer Research, 16, 620
(1956).

4. M. L. Petermann and M. G. Hamil-
ton, /. Biol. Chem., 224, 725 (1957).

5. N. Simmons, private communication.

6. L. Mandelkern and P. J. Flory,
/. Chem. Phys., 20, 212 (1952).

7. P. J. Flory, Principles of Polymer
Chemistry, p. 622, Cornell University Press,
Ithaca, N. Y., 1953.

8. R. Thomas, Biochim. ct Biophys.
Acta, 14, 231 (1954).

9. B. Magasanik in The Nucleic Acids,
vol. 1, p. 393, Academic Press, New York,
1955.

Microsomes and Ribonucleoprotein Particles

GEORGE E. PALADE

The Laboratories of the Rockefeller Institute for Medical Research

I should like to present in the following pages a short history of the develop-
ment of our present concepts on microsomes and ribonucleoprotein (RNP)
particles. I consider the historical background of this field of research interesting
in itself; moreover, I believe that its knowledge may throw some light on the
actual relations between microsomes and RNP particles as well as on some basic
principles of biological organization.

THE MICROSOMES

The discovery of the microsomes was a by-product of work done on virus-
induced tumors by Albert Claude at the Rockefeller Institute in the late 1930's.
Trying to purify a tumor-inducing fraction obtained by differential centrifuga-
tion from breis of Rous sarcomata,1 Claude found as expected that the prepara-
tion was rich in ribonucleic acid (RNA) [1], and was inactivated by various
agents known to affect nucleoproteins and nucleic acids [2]. At the same time,
however, he unexpectedly discovered that cell fractions, similar in their gross
chemistry to the tumor-inducing preparations, could be isolated from chick
embryos [3], and from a variety of tissues, adult as well as embryonic, and
normal as well as tumorous [4, 5]. From the beginning Claude was convinced
that these fractions consisted of pre-existing cell structures, not of cytoplasmic
aggregates artificially produced by tissue grinding. After some hesitation,2 he
[4, 5] arrived at the conclusion that the structures involved were new cell
components of widespread occurrence which had eluded detection by light
microscopy because they were too close to, or below, the limit of resolution of

1 Chicken tumor I.

2 For a while he assumed that the fraction consisted of mitochondria or mitochondrial
fragments.

PALADE 37

usual light optics.3 In his early reports he described the new components as
"small particles" or "small granules"; later he chose the term "microsomes"
(small bodies) [6, 7], which met with favor and has remained in common use
ever since. From his centrifugation data,4 Claude calculated that the micro-
somes measured ~ 50 to 200 m/* in diameter, and on the strength of his
chemical analyses he defined them as "phospholipide-ribonucleoprotein com-
plexes" [4, 5]. In this way and thus defined the microsomes entered the bio-
chemical thinking of our times.

Claude's discovery was followed by an extensive period of biochemical re-
search which confirmed and greatly extended his findings. For practical rea-
sons, the research effort was concentrated almost exclusively on liver, and as a
result the voluminous literature thereby produced (for reviews see [8, 9, 10])
applies primarily to liver microsomes, not, as usually assumed, to microsomes in
general. According to this literature, the dominant biochemical feature of the
microsomal fraction is its high RNA content: ~ 40 to 50 per cent of the RNA
of the tissue brei is usually recovered in the microsomes, together with ~ 15 per
cent of its proteins. Consequently the microsomal RNA/protein ratio is high—
until recently higher than that of any other cell fraction. It should be men-
tioned, however, that, with all this concentration, the RNA does not represent
more than ~ 10 per cent of the microsomal dry weight. Another apparently
characteristic feature of the fraction is its large content and high concentration
of phospholipides : ~ 50 per cent of the phospholipides of the tissue are re-
covered in the microsomes. As far as biochemical activities are concerned, the
microsomes are distinguished by high concentrations of diphosphopyridine
nucleotide-cytochrome c reductase [11], cytochrome m or b-0 [12, 13], and glu-
cose-6-phosphatase [14], and especially by their ability to incorporate labeled
amino acids into their proteins both in vivo [15-19] and in vitro [20-22]. Ac-
cording to current interpretations, the last property indicates that the micro-
somes are, or contain, the sites of protein synthesis of the cytoplasm.

In contrast with the active and diversified work on the biochemical aspects
of the problem, research on the identity of the microsomes, or of their pre-
cursors, inside the intact cell made little progress because of a number of tech-
nical limitations. Of the instruments available for morphological investigation,

3 The microsomes can be seen as distinct particles in the dark-field microscope [5], and
as a shimmering mass of indistinct small bodies in the light microscope, especially under
phase contrast optics.

4 For Claude the microsomes were the fraction sedimented in 1 hour at 18,0(% from the
supernatant of the "large granule" (mitochondrial) fraction. The medium he used in pre-
paring tissue "extracts" was either water or dilute phosphate buffer or 0.15 M NaCl. When
sucrose solutions were introduced in cell-fractionation procedures, the centrifugal force was
increased to compensate for the higher density and viscosity of the new medium. At
present the microsomes are usually separated by centrifuging a mitochondrial supernatant
for 1 hour at ~ 100,00%, irrespective of the sucrose concentration in the suspending
medium. Since this concentration varies from 0.25 to 0.88 M, the microsome fractions
described in the literature are not strictly comparable to one another.

38 MICROSOMAL PARTICLES

one — the light microscope — did not have enough resolving power, and the other
— the electron microscope, introduced in biological research at the time of the
discovery of the microsomes — could provide the necessary resolution but pre-
paratory techniques for the electron microscopy of biological specimens were
still inadequate. As a result the microsomes remained until recently a cyto-
chemical concept only, without a known structural counterpart in the organiza-
tion of the intact cell. Many cytologists and cytochemists even doubted that
these small bodies were derived from a pre-existing cell structure and were
inclined to consider them artifacts due to cytoplasmic clumping or mitochon-
drial fragmentation [23] during the homogenization of the tissue.

Because of their characteristically high content of RNA, the microsomes were
rather early correlated with the so-called "basophil substance" of the cytoplasm.
Although the correlation was based on circumstantial evidence obtained on a
limited number of cell types [7, 24], it was soon assumed to be generally valid,
and consequently the microsomes and the basophil cytoplasm began to be re-
garded as equivalent terms in two different technical vocabularies. An apparent
convergence was thus effected between two lines of cytochemical research: an
old line exemplified by the work of Brachet's (cf. [25]) and Cassperson's (cf.
[26]) groups and aiming at the localization of nucleic acids in situ, and a new
line based on cell-fractionation procedures. The correlation appeared to be
further strengthened when the large body of circumstantial evidence accumu-
lated by Brachet and Cassperson on the role of RNA in protein synthesis
received full and repeated support from experiments showing that the micro-
somes were the most active cell fraction in the incorporation of labeled amino
acids into proteins.

The lack of morphological information, the concentration of the work on
liver, and the equation of the microsomes with the basophil substance of the
cytoplasm had some unfavorable consequences on the development of general
ideas in the field. It was assumed, for instance, without enough evidence, that
entities similar or identical to Claude's microsomes existed in all cells, bacterial
cells included, and it was also believed that the chemical composition of liver
microsomes was representative for microsomes in general. As we shall see, both
assumptions are now in need of revision.

THE ENDOPLASMIC RETICULUM

I shall turn now to another development, in a different field, which from the
beginning seemed to have some connection with our main problem. A few
years after the discovery of the microsomes, and again in conjunction with work
done on Rous sarcomata, Porter, Claude, and Fullam \27] and Claude, Porter,
and Pickels [28] succeeded in obtaining electron micrographs of thinly spread
cells (avian fibroblasts) maintained in tissue culture. Below the limit of reso-
lution of the light microscope and well within the range of calculated micro-
somal sizes, they found a "lace-like" network of slightly higher density than the

PALADE 39

rest of the cytoplasm. Porter, continuing the study of this structure in cultured
material, arrived at the conclusion that it consisted of vesicles and tubules
interconnected in a continuous network. Since the network was restricted to
the inner or endoplasmic region of the cytoplasm, he proposed the name endo-
plasmic reticulum for the entire structure [29, 30]. The discovery of this net-
work provided a likely candidate for the role of microsomal precursor within
the intact cell, and indeed both Claude [31] and Porter [30] speculated that
the microsomes might represent derivatives of the endoplasmic reticulum, but
the assumption could not be verified at the time because the electron microscopy
of cells and tissues was still in infancy. The examination of a liver cell, for
instance, was a difficult and uncertain project; in fact, the first attempt [32]
to identity "microsomes" in electron micrographs of sectioned hepatic cells
yielded misleading results.5

In the early 1950's, however, a succession of technical improvements covering
the whole series of preparative steps [33, 34] but affecting primarily microtomy
[35, 36] made possible the examination of thin sections of practically all cell
types in the electron microscope. With this spectacular breaking through the
barriers of technical difficulties, the search for the intracellular equivalents of
the microsomes finally became possible. But most electron microscopists en-
gaged at that time in the study of the fine structure of the cell were not directly
interested in the microsome problem; they were rather attracted by a related
question: namely that of the structural substrate of cytoplasmic basophilia.
Apparently it did not matter, because, as already mentioned, the two problems
were expected to have a common solution. In the newly opened realm of fine
cellular organization the microscopists found an unsuspected and, at the begin-
ning, puzzling abundance of structures. After a few years spent in deciphering
these findings and in arguing about various interpretations,6 it became clear
that the ground substance of the cytoplasm contained an extensive system of
spaces, described as vesicles, tubules, and cisternae 7 (figs. 1 and 2), limited by
a thin membrane (~ 7 m/*) and interconnected in a more or less continuous
network.

It was soon realized that the so-called ground substance of the cytoplasm was
divided into two distinct phases by the existence of this internal membrane
system: one represented by the content of the interconnected vesicles, the
other by the surrounding cytoplasmic matrix. It was also observed that small,
dense particles, ~ 15 m/x in diameter, appeared to be attached to the membrane
(on the surface facing the cytoplasmic matrix) in certain parts of the reticulum
(fig. 2) while other parts remained free. In addition, it was found that similar
particles occurred apparently freely scattered throughout the cytoplasmic matrix

r' Masses of glycogen were apparently taken for microsomes.

6 Representative samples of the various interpretations advanced can be found in refer-
ences 37 to 39.

7 The term designates flat, shallow vesicles which measure only 50 to 70 m(i in depth
but reach into microns in the other two directions.

40 MICROSOMAL PARTICLES

(fig. 2). Work done in our laboratory established that the system corresponds
to the endoplasmic reticulum of cultured cells [40], and subsequent observa-
tions showed that a number of local differentiations occur within this con-
tinuous network which appears to possess, for instance, a rough-surfaced part
[40, 41], on account of the attached particles already described, and a smooth-
surfaced [40, 41] or agranular [42] part free of such particles. Frequently the
elements of the system, particularly its cisternae, show preferred orientation
and seem to be disposed parallel to one another at more or less regular intervals,
thus forming stacks or piles of various sizes (figs. 1 and 2). Finally, further w.vrk
showed that the system varies characteristically from one cell type to another
and that these variations affect the total volume of the system, the relative extent
of its rough- and smooth-surfaced parts, as well as the extent of preferential
orientation encountered within the system [38].

The Structural Substrate of Cytoplasmic Basophilia. With the limited infor-
mation initially available, it was believed that the endoplasmic reticulum as a
whole was the structural substrate of basophilia, but subsequent observations
brought forward serious discrepancies between the distribution of the reticulum
on one side and that of basophilia on the other. It was found, for instance, that
there are cell types with an intensely basophil cytoplasm in which the endo-
plasmic reticulum is poorly developed. Such cells, however, have a large popu-
lation of small, dense particles, most of them freely scattered in the cytoplasm
[43]. The erythroblasts and the undifferentiated cells of rapidly growing
epithelia (epithelia of the intestinal crypts, stratum germinativum of the epi-
dermis) belong to this category. A converse situation is encountered in mature
leucocytes and in seminal epithelia (rat) whose acidophil cytoplasm contains
a relatively well developed endoplasmic reticulum, most of it smooth-surfaced,
but has only a few small particles, free or attached.

In all the cases examined, the cytoplasmic component whose distribution
matched best that of the affinity for basic dyes appeared to be represented by the
small, dense particles. As a result of these findings, in 1953 I advanced [44, 43]
the hypothesis that these particles, rather than the membranous material of the
endoplasmic reticulum, contained most of the RNA of the cytoplasm, and that
they were consequently the sought-for structural substrate of cytoplasmic baso-
philia. The hypothesis rested upon the results of a broad survey of various cell
types which covered a large number of "test specimens," i.e., cells known for
the intensity and characteristic distribution of "basophil substance" in their
cytoplasm, as well as cells known for their cytoplasmic acidophilia. The postu-
late derived additional support from the fact that at that time small particles in
the same size range and containing a large amount of RNA had already been
isolated from yeast and bacterial cells [45] by Schachman et al., and from certain
mammalian tissues, such as liver and spleen, by Petermann et. al. [46, 47].

From the beginning the hypothesis implied that the small particles and the
endoplasmic reticulum represent two basically distinct components of the cyto-
plasm which may exist and develop independently of each other. Their close

PALADE 41

and more or less extensive association was considered a secondary phenomenon
that occurs at a relatively late stage in the evolution of cellular organization.
The view, originally based on findings on undifferentiated or embryonic cells,
subsequently received full support from electron-microscope studies of various
bacteria [48, 49, 50] which revealed that bacterial protoplasm contains a large
population of small, dense particles (usually smaller than those found in animal
cytoplasm), but apparently no internal membranous system comparable to the
endoplasmic reticulum [50].

A Correlated Morphological and Biochemical Analysis of Hepatic Micro-
somes. The next task was to put the hypothesis to a test by trying to find out in
what cell fractions the particles segregate during the differential centrifugation
of tissue breis. Fraction chemistry and particle distribution were correlated by
using duplicate pellets of the fractions under study : one pellet for biochemical
analysis, and the other for electron microscopy after appropriate fixation, em-
bedding, and sectioning. It was found both necessary and expeditious to fix the
pellets in toto, and to cut them in such a way as to be able to survey them from
top to bottom. With such precautions, the existence and the extent of inter-
contamination among cell fractions could be easily detected and the presence of
distinct layers in some pellets clearly demonstrated.

The work, carried out in collaboration with Dr. Philip Siekevitz, started
with an analysis of the microsomal fraction isolated from rat-liver breis [51].
We found that this fraction consists almost exclusively of closed vesicles limited
by a dense continuous membrane, ~ 7 mp thick, and filled with a material of
relatively low density. Most of these vesicles are derived from the rough-surfaced
part of the endoplasmic reticulum as indicated by the small (~ 15 mp), dense
particles attached to the outer surface of their limiting membrane. Smooth-
surfaced vesicles are also present in the microsomal fraction, but their origin is
more difficult to ascertain; they may represent fragments of the smooth-surfaced
part of the reticulum or they may be derived from other sources (Golgi com-
plex? 8 cell membrane?). In 0.88 M sucrose, the medium used in our experi-
ments, the microsomal vesicles retained the flattened appearance of intracellular
cisternae and reacted like osmometers to changes in the concentration of the
medium: they swelled in hypotonic media. Treatment with versene (2 per cent
in 0.88 M sucrose, for 60 minutes at 0° C) removed ~ 60 per cent of the micro-
somal RNA and resulted in extensive loss of attached particles. Incubation in
ribonuclease (0.5 mg/ml 0.88 M sucrose; 60 minutes at 37° C) caused RNA
losses of ~ 85 per cent and produced a heavy agglutination of microsomal
vesicles. During the incubation, the attached particles apparently were lost.
Finally treatment with sodium deoxycholate (DOC) (0.5 per cent in 0.88 M
sucrose, at pH 7.5) "solubilized" most of the protein and phospholipides of the

8 According to our interpretation [38], the "Golgi complex" is a differentiated part of
the endoplasmic reticulum. Other cytologists [52] consider this structure a distinct and
independent cell organelle.

42 MICROSOMAL PARTICLES

microsomes, but left ~ 80 per cent of their RNA and ~ 15 per cent of their
protein still in sedimentable form. The pellets obtained from DOC-treated
microsomes consisted of small, dense particles ~ 10 to 15 rw* in diameter with
a small admixture of vesicles. We interpreted the results as indicating that
DOC "solubilizes" the membrane and content of the microsomal vesicles while
affecting their attached particles to a lesser extent, and we inferred from these
experiments that the small, dense particles consist of ribonucleoprotein and con-
tain most of the microsomal RNA (~ 80 per cent), while the membrane and
content account for most of the protein and almost all the phospholipides of
the microsomes [51].

We soon found out that, contrary to assumptions then current, the final
supernatant still contained numerous structured elements of membranous or
particulate nature, and accordingly we made an attempt to separate these ele-
ments by further centrifugation of the microsomal supernatant, the fraction
usually considered the liquid phase of the cytoplasm or the "cell sap." We iso-
lated two successive postmicrosomal fractions ° in the hope that one of them
might consist mainly of free particles, but we found the corresponding pellets
to be mixtures of smooth-surfaced vesicles (probably derived from the smooth-
surfaced part of the reticulum), free particles, and amorphous material. The
RNA content of these postmicrosomal fractions was low, and its concentration
therein considerably lower than in the microsomes [51].

In the case of the liver we succeeded, therefore, in identifying the microsomes
as fragments of the endoplasmic reticulum, derived primarily from its rough-
surfaced part. We obtained evidence indicating that they are closed vesicles,
and that most of their RNA is present as ribonucleoprotein in their attached
particles. We did not succeed in isolating the free particles of the cytoplasmic
matrix, nor did we obtain a "clean" preparation of smooth-surfaced vesicles. The
information on the gross chemistry of microsomal membranes was indirect and
relied on subtractions and many assumptions.

PANCREATIC MICROSOMAL AND POSTMICROSOMAL FRACTIONS

As the next object for testing our hypothesis, we chose the pancreas of the
guinea pig with the following considerations in mind. The exocrine cells,
which form the bulk of the cell population of the gland, have an endoplasmic
reticulum remarkable in its large volume and in the small extent of its smooth-
surfaced part (figs. 1 and 2). In addition, their cytoplasmic matrix contains free
particles in great numbers (fig. 2). With the new material, therefore, we stood
a better chance to obtain a more homogeneous microsomal fraction and to
separate free particles in a postmicrosomal fraction. The results met our expecta-

9 The first postmicrosomal fraction (PMj) was obtained by centrifuging the microsomal
supernatant for 2 hours at 105,000^; the second postmicrosomal fraction (PM2), by
centrifuging the supernatant of PM: for 16 hours at 105,000 g.

PALADE 43

tions. We found that pancreatic microsomes [53] are closed vesicles (figs. 4
and 5) almost exclusively derived from the rough-surfaced part of the cells'
voluminous reticula. They are, however, more labile structures than their
hepatic counterparts: even when isolated in 0.88 M sucrose they enspherulate
and do not retain the flattened cisternal form that the elements of the endo-
plasmic reticulum have in situ. They are also more susceptible to various treat-
ments, especially to deoxycholate, which, at 0.3 per cent final concentration,
solubilizes ~ 85 per cent of their protein and ~ 40 per cent of their RNA. As
in the case of the liver, the DOC-insoluble material consists of RNA particles
(fig. 6) with a relatively high RNA/ protein ratio. The main difference be-
tween hepatic and pancreatic microsomes concerns their phospholipide content:
there is ~ 8 times less phospholipide in pancreatic microsomes than in their
hepatic counterparts, and the phospholipide concentration in the microsomal
fraction is equal to, or only slightly higher than, that in the original pancreatic
brei.

Centrifugation of the microsomal supernatant resulted in the sedimentation of
further material which was arbitrarily divided into two postmicrosomal frac-
tions °( PMi and PM2). We found that the corresponding pellets consist almost
exclusively of small, dense particles ~ 15 m^ in diameter (fig. 7), with a mod-
erate admixture of small vesicles in the PMi. Chemically both fractions were
made up of ribonucleoproteins with a negligible amount of phospholipides.
In the original fractionation scheme [53], the two postmicrosomal fractions
contained RNA in comparable concentrations, but in a recent modification
PMi has a noticeably higher RNA/protein ratio than PM2.

The results obtained with the pancreas confirmed and extended our previous
findings on liver. In both the microsomes were found to be vesicles derived
either to a large extent [51], or almost exclusively [53], from the rough-surfaced
part of the endoplasmic reticulum. We used a characteristic structural detail,
namely the attached particles, to establish this derivation, and our conclusion
appears reasonably valid; there is good general agreement between the basic
morphological features of the microsomes and the fine structural details of the
endoplasmic reticulum. There is, however, no agreement as far as general dimen-
sions are concerned: the network is a continuous structure which may spread
throughout the entire cytoplasm of the intact cell (~ 20 X ~ 30 X ~ 40 fi),
whereas the microsomes are considerably smaller (0.05 to 0.3 n), corresponding
in dimensions to the vesicles, tubules, and cisternae which by their interconnec-
tions form the reticulum. Accordingly we must assume that the network is
broken during tissue grinding, the resulting fragments being the microsomes.
Since these fragments are closed vesicles, we are further obliged to postulate that
the broken segments heal readily into closed structures or that the fragmenta-
tion of the reticulum occurs by a generalized pinching-ofT process rather than
by mechanical disruption [51, 53].

44 MICROSOMAL PARTICLES

DIVERSITY OF CYTOPLASMIC RIBONUCLEOPROTEINS

The view that the small, dense particles represent the structural substrate of
cytoplasmic basophilia was clearly supported by the isolation of RNP particles
from DOC-treated microsomes (fig. 6) and further strengthened by the finding
that the pancreatic postmicrosomal fractions (fig. 7), assumed to represent the
free particles of the cytoplasmic matrix, are also comprised of ribonucleopro-
teins. It should be pointed out, however, that the RNP particles, free and at-
tached, do not account for all the RNA of the cytoplasm. Leaving aside the
controverted question of the RNA content of mitochondria, we found that the
microsomes contain a certain amount of RNA, small in the liver but relatively
large in the pancreas, which is solubilized by DOC treatment, under the condi-
tions of our experiments, and whose structural connections are unknown. Fi-
nally there is a relatively small amount of RNA (~ 10 per cent of the RNA of
the cytoplasm) that remains in the supernatant of the last postmicrosomal frac-
tion. Evidently we do not know to what extent our findings reflect the situa-
tion inside the living cell, or to what extent they are affected by preparation
artifacts. It can be argued, for instance, that attached particles can be detached
during tissue grinding and fractionation, or that the sedimentation of RNA
particles is still incomplete by the end of our last centrifugation.

With such possibilities in mind, Dr. Siekevitz and I tried to find out in more
recent work [54] whether there are functional differences among the various
RNP preparations described. In agreement with reports on other tissues [55,
56], we noted that the attached particles (DOC-insoluble microsomal material)
are more active than the parental microsomes in the incorporation of labeled
amino acids into proteins (presumably protein synthesis10). In addition, we
found that the activity mentioned is considerably higher in the attached particles
than in the free RNP particles of the postmicrosomal fractions. The second
postmicrosomal fraction, however, proved to be more active than any other
particulate fraction in the incorporation of labeled adenine into RNA. In turn,
its activity was greatly exceeded by that of the "soluble" RNA of the final
supernatant.

Such metabolic differences, though not entirely excluding preparation arti-
facts, render them more unlikely, and suggest that a variety of RNP's with dif-
ferent structural connections and different functions exist within the animal
cell. I should also add that by comparing microsomes obtained from starved
and fed animals we observed a relatively large increase in microsomal pro-
teolytic11 and ribonuclease activity [57]. What seems to be of considerable
interest is the finding that a sizable part of the enzymatic activities of the micro-

10 In our experiments the incorporation was carried out in vivo; in those reported in
references 55 and 56 the labeled amino acids were incorporated either in vivo or in vitro
by whole microsomes.

11 Due mainly to trypsinogen and chymotrypsinogen. These two proteases and the
ribonuclease are enzymes synthesized on a large scale by the exocrine cells of the pancreas
to be released in the intestine for the digestion of food.

PALADE 45

somes is found associated with their attached RNP particles. Further work will
show whether these enzymes are newly synthesized proteins still attached to
their sites of synthesis or enzymes released from other locations and absorbed
on RNP particles during tissue grinding.

DIFFERENCES BETWEEN MICROSOMES AND RNP PARTICLES

It follows from the results thus far summarized that the cytoplasm contains
more separable entities than were assumed a few years ago. It also follows that
Claude's microsomes are relatively large and complex structures in which mem-
branous and particulate components can be easily recognized. There is also a
microsomal content which, though usually amorphous, may occur as formed
granules ([57], cf. [58]) under certain conditions, thus increasing the com-
plexity of the structure. The term "microsomal particles," used in many com-
munications made at this meeting, can be properly applied to RNP particles
attached to the surface of microsomal vesicles. As already indicated, such par-
ticles can be detached by DOC treatment and subsequently collected in rela-
tively clean preparations. It is doubtful, however, that the same term can be
used to designate the free RNP particles isolated in postmicrosomal fractions
and especially RNP particles separated from bacterial cells, in which there is
no endoplasmic reticulum to start with, and from which no microsomes can be
obtained. Pellets obtained from bacteria are composed of much smaller and
simpler cell components which morphologically seem to correspond to the free
RNP particles of the pancreatic cell. In general, a morphological label is justi-
fied as long as there is no information on the chemistry and function of the
structure involved. Here, however, we know that we are dealing with ribonu-
cleoprotein particles, and consequently there is not too much sense in retaining
a morphological label, especially after realizing that it is misleading.

More than accurate terminology is involved in this argument. What is known
so far about the fine structure of bacterial cells suggests that internal mem-
branous systems, like the endoplasmic reticulum, are not necessary for the
organization and function of a simple type of self-sustaining cell. Such mem-
branous systems appear in more elaborate cell forms and could therefore be
regarded as superstructures. We do not know what special problems are solved
by their introduction, but we may wonder whether they are not connected with
an increase in cell volume, subsequent difficulties in diffusion, and relative
decrease in available surface. At higher levels of biological organization, similar
problems are frequently encountered and usually solved by the invagination of
surface structures and by the concomitant interiorization of part of the sur-
rounding medium. There is therefore an important difference in organization
between bacterial cells on one side, and animal and plant cells on the other:
the superstructure that can be ground into microsomes appears only in the
latter. Terms such as "microsomes" or "microsomal particles" of bacterial
origin do not take this basic difference into account.

46 MICROSOMAL PARTICLES

RECENT INFORMATION ON CYTOPLASMIC BASOPHILIA AND RNP PARTICLES

During the past 5 years considerable progress has been made in the study of
cell organization through the extensive use of electron microscopy and cell-
fractionation procedures. Accordingly it is of interest to see how the hypothesis
formulated in 1953 has fared through this period of rapid development.

The assumption that the small particulate component of the cytoplasm is
the structural substrate of basophilia has remained in good agreement with
the large majority of the findings made on new and very numerous cell types
of animal and plant origin (see for examples [59, 60]). The only exception so
far encountered is represented by the heart muscle of the turtle [61], in which
a slightly larger particulate was found in an acidophil cytoplasm that gave a
positive test for glycogen. Consequently it was postulated that particulate glyco-
gen might be mistaken for RNP particles in certain cell types, especially in
muscle [61]. The actual isolation of this particulate material from various
muscle fibers could settle the question, but so far it has not been accomplished.
It should be pointed out, however, that under prevailing technical conditions a
certain amount of confusion of the type suggested cannot be excluded. Because
of the dimensions involved, we are examining only the gross morphology of
the small particles, and in so doing we are not helped thus far by any charac-
teristic detail of structure. If particles of different chemical composition and of
different fractions happen to have the same general size and shape, we cannot
avoid lumping them together in a common category. Morphological expressions
that can be distinguished at the present level of practical resolution are un-
doubtedly less numerous than functional characteristics or macromolecular
species. It is exactly for this reason that morphological information should be
supplemented, wherever possible, by biochemical and metabolic data.

Ribonucleoprotein particles, with a sedimentation constant of 70 to 80 S and
a calculated or measured diameter of 10 to 15 m/x, have been isolated from
many new and old sources such as liver [62, 65], yeast [63], ascites cells [56],
and pea seedlings [64]. They have been described in terms of their gross
chemistry [62-65], biochemical activities [56], and physicochemical properties
[62-65]. A perusal of this symposium shows that recently RNP particles have
also been isolated from a variety of bacterial cells. In general, there is good
agreement between these findings and our observations, but there is little or no
information about the existence, frequency, and topography of the particles
in situ.

Finally it is still debated whether the microsomal RNA is mainly located in
the attached particles or is also present in large amounts in the microsomal
membranes as originally assumed by Kuff et al. [66]. As already mentioned,
an exclusive RNA location in the attached particles cannot be claimed because
the particles do not account for ~ 20 per cent of the microsomal RNA in the
liver and for ~40 per cent in the pancreas. Recently Chauveau et al. [67]
found that there is no good correlation between the frequency of vesicles with

PALADE 47

attached particles and the amount of RNA present in microsomal pellets. They
indicate, however, that particles appear after treating predominantly mem-
branous pellets with DOC, and they suggest that the particles could be masked
by incorporation into the membrane under certain uncontrolled conditions. It
is evident that we should know more about the behavior of these particles
under various metabolic conditions, and about their reaction to various suspen-
sion media used in tissue fractionation, before arriving at an understanding
of these conflicting pieces of evidence.

CONCLUSIONS AND COMMENTS

In conclusion it can be said that small (~ 15 m/*), dense particles have been
found, either free or attached to the membrane of the endoplasmic reticulum,
in practically all animal and plant cells thus far examined. Comparable parti-
cles, usually unattached to membranous structures, exist in bacterial cells. The
distribution of these particles is largely similar to that of cytoplasmic basophilia,
by implication to that of cytoplasmic RNA.

Ribonucleoprotein particles of relatively small size (10 to 20 mn and 40 to
80 S) have been isolated by various procedures, such as ultracentrifugation and
electrophoresis, from a variety of animal, plant, and bacterial sources. Evidence
that the small, dense particles seen in the intact cell consist of ribonucleoprotein
has been obtained for the liver (rat) and the pancreas (guinea pig). Accord-
ingly the assumption that these particles are the structural substrate of basophilia
has been verified for two cell types only. For all the others, it remains what
it has been, a hypothesis to be tested by further work. Integrated studies pro-
viding an adequate coverage from cell to pellets are evidently needed for more
kinds of tissues.

Although it appears that the RNP particles are cytoplasmic components of
widespread occurrence, probably basic structural elements in the organization
of the cell, many points in their history are still uncertain or controverted, and
many pertinent questions remain unanswered. For instance, though there is
good agreement about the presence of RNA in particles, there is still doubt
about the presence or absence of RNA in membranous structures, primarily in
the membrane of the endoplasmic reticulum. Considerable variation occurs,
apparently connected with the methods of preparation, in the RNA content of
these particles; accordingly one would like to know the procedure by which
the situation in situ is more closely approximated. There is morphological,
physicochemical, and metabolic diversity among these particles, but very little
is known about the way in which the various differences are correlated, or
about the significance of this diversity. In this respect one may wonder whether
particles with different locations and activities represent distinct, fully developed
cell organs, or whether they correspond to successive stages in the differentiation
of a single or a few cell organs. What seems to be particularly disturbing at
present is the meagerness of our information about the functional role of these

MICROSOMAL PARTICLES

particles. Without more knowledge on this aspect, progress is uncertain even
if it takes the appearance of elegant physicochemical data.

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Fig. 1. The electron micrograph shows part of two adjacent exocrine cells in the
pancreas of a guinea pig. The apposed membranes of the two cells appear obliquely sec-
tioned at cm.

The basal region of one of these cells occupies the lower half of the figure and char-
acteristically contains a few mitochondrial profiles (m) and numerous profiles of the
endoplasmic reticulum (rs), which belong to the rough-surfaced type and show a certain
amount of preferred orientation, i.e., are disposed in more or less parallel rows.

The apical region of the cell, in the upper part of the figure, is occupied by a few circular
profiles of zymogen granules (z) . Their dense content consists of stored digestive enzymes
and enzyme precursors. Elements of the endoplasmic reticulum appear disposed at random
among the zymogen granules. Part of the centrosphere region of the cell, with its char-
acteristic clusters of smooth-surfaced vesicles, can be seen at cs.

A region similar to the lower right quarter of this figure appears in figure 2 at a higher
magnification.

Specimen fixed for 2 hours at 0° C in 1 per cent Os04 in veronal acetate buffer, pU 7.6,
containing ~5 per cent sucrose; embedded in n butyl methacrylate.

Magnification: 24,000.

Fig. 2. The micrograph shows at a high magnification a small field in the basal region
of a pancreatic exocrine cell (rat).

Parts of two mitochondrial profiles appear at m; the rest of the field is taken by numerous
profiles of the endoplasmic reticulum (rs), most of which are of elongated form and appear
disposed in parallel rows. In three dimensions many of these profiles correspond to rela-
tively large but flat vesicles known as cisternae.

The membrane limiting the cavities of the reticulum appears as a dense, fine line (n)
whenever sectioned normally, and as a less dense, poorly outlined band (o) when cut
obliquely. In a few places the section has opened small windows (/) in the wall of the
cisternae.

The membrane of the endoplasmic reticulum separates two distinct phases in the
cytoplasm: one is represented by the light, homogeneous material enclosed in the cavities
of the system (c); the other, by the surrounding cytoplasmic matrix (mx).

Numerous small, dense particles, — 150 A in diameter, appear attached to the outer
surface of the membrane limiting the cavities of the endoplasmic reticulum (ap). In ad-
dition to these attached particles, particles of comparable size and density occur apparently
freely scattered in the cytoplasmic matrix (fp). Note, however, that many of these particles
form short chains (arrows) anchored with one end among the attached particles.

Fixation: 24 hours at room temperature in 1 per cent Os04 in acetate veronal buffer,
pH 7.6. As a result of the long fixation, part of the cytoplasmic matrix has been extracted
and the profiles of the endoplasmic reticulum and the small, dense particles appear in
better contrast.

Embedding: n butyl methacrylate.

Magnification: 50,000.

•N.

SMHT

■

H :

I _<>

Fig. 3. The micrograph shows a small field in the basal region of a pancreatic exocrine
cell which was damaged (cut open) during the trimming of the tissue block before fixation.

A comparison with figure 2 indicates that in the damaged cell the profiles of the endo-
plasmic reticulum have distended cavities and are predominantly circular. In three dimen-
sions they correspond to spherical and oval vesicles. An enspherulation of this type is
usually accompanied by a breaking-down of the system into a collection of isolated vesicles.
Note that the vesicles are still aligned in more or less parallel rows.

The limiting membrane of the vesicles and its attached particles appear in normal section
at n, and in oblique section at o. In this case there are few free particles left in the
cytoplasmic matrix.

Fixation and embedding as for the specimen in figure 2.

Magnification: 50,000.

l ti.

Fig. 4. Section through a microsome pellet isolated by differential centrifugation from a
pancreatic brei (guinea pig) prepared in 0.88 M sucrose.

The microsomes are small, closed vesicles limited by a thin membrane which bears small
(~150 A), dense particles attached to its outer surface. The apparent heterogeneity of the
microsomal fraction is due primarily to sectioning. Some vesicles are seen in median sec-
tion {mvx) and therefore clearly display their normally sectioned membrane and their
attached particles. Other vesicles are cut medially (mvz) and show obliquely sectioned,
poorly defined membranes. Finally, in lateral sections (mvs), the cavity of the microsomes
cannot be seen, and their particle-studded membrane appears in full-face view.

The structural details described indicate that the microsomes are derived (by fragmenta-
tion) from the rough-surfaced part of the endoplasmic reticulum. A comparison with
figure 2 suggests that the fragmentation occurs spontaneously when the cell membrane
is ruptured during tissue grinding.

Note that the microsomal content varies widely in density from light (rhv±) to medium
(raz/4) and high (mi/5). A ruptured microsomal vesicle (mvG) contains the equivalent of
an intracisternal granule.

Fixation: 2 hours at 0° C in 2 per cent Os04 in 30 per cent (0.88 M) sucrose.

Embedding: n butyl methacrylate.

Magnification: 72,000.

Fig. 5. Small field in a microsomal pellet (guinea pig) prepared like die specimen in
figure 4.

A few microsomal vesicles appear in median {mv^), medial (mv2), and lateral (mv3)
section. The particles attached to the outer surface of the limiting membrane (arrows)
display their characteristic small size ( — 150 A) and high density.

Fig. 6. Small field in a pellet obtained by differentially centrifuging a microsomal
suspension (like the one in figure 5) treated with Na deoxycholate (0.1 per cent, pH 7.2).

The pellet consists of dense particles, — 150 A in diameter, which frequently occur in
chains (arrows) or in clusters. Their general morphology suggests that they are particles
detached from the microsomes as a result of the solubilization of the microsomal membrane
by deoxycholate.

Both pellets were fixed and embedded like the specimen in figure 4.
Magnification: 120,000 for both figures.

Fig. 7. Pellet of a second postmicrosomal fraction obtained by differential centrifugation
(16 hours at 105,000^) from a pancreatic brei (guinea pig) prepared in 0.88 M sucrose.

The fraction consists of small (~150 A), dense particles which occur either isolated or
in chains (arrows) . They are assumed to be the "free" particles of the cytoplasmic matrix.

Fixation and embedding as for the specimen in figure 4.

Magnification: 160,000.

*»

The Influence of Conditions of Culture on
Certain Soluble Macromolecular Components

of Escherichia colt

S. DAGLEY J- SYKES

Department of Biochemistry, University of Leeds, England

Schachman, Pardee, and Stanier [1] used the analytical ultracentrifuge to
examine soluble extracts prepared from various bacterial species disrupted by
different methods, and they showed the presence in all extracts of three major
components having sedimentation coefficients (uncorrected) of about 40, 29,
and 5 S. Other workers (Siegel, Singer, and Wildman [2] and Billen and
Volkin [3]) have obtained ultracentrifuge patterns in substantial agreement.
For the strain of Escherichia coli we have used, a typical "basic" pattern is seen
in figure 3a, where the boundaries, reading from left to right, sediment re-
spectively at 40, 29, and 20 S followed by a large, slow-moving peak which, on
centrifuging for longer periods, resolves into two peaks of 8 and 5 S. Although
the ultracentrifuge has revealed a common pattern of macromolecules, how-
ever, the ever-increasing range of enzymes shown to be induced in bacteria
supports the assertion that they are "the most plastic of living material" (Ste-
phenson [4]); and accordingly we have tried to find out whether changes in
their environment have any influence upon the ultracentrifuge pattern.

This work may be divided into two parts. First, we examined the basic ultra-
centrifuge pattern for modifications that might result from growth of the cells
on different sources of carbon; and we also determined rates of sedimentation
of certain enzymes, some of which were induced by addition of substrates to
cultures, in order to decide whether any of them appeared to be associated with
macromolecules revealed by ultracentrifugal analysis. The second series of
experiments was concerned with factors affecting the concentration of the 40 S

component inside the living cells.

DAGLEY AND SYKES 63

SEDIMENTATION OF ENZYMES

E. coli cells were usually grown without aeration in media containing 0.13 M
KH2PO4 brought to pH 7 with NaOH; growth in 0.01 M glucose was then
limited entirely by exhaustion of the source of carbon, and changes in pH
were negligible. The medium was completed by addition, per liter, of 0.2 g
MgSOWHoO and either 1 g (NH4)2S04 (for a "mineral salts" medium) or
10 g Difco bactopeptone. Extracts were prepared from cells disintegrated in
the Hughes [5] bacterial press without abrasive; soluble material was ex-
tracted by stirring with 0.066 M phosphate buffer, pH 7, and cell debris was re-
moved by centrifuging for 15 minutes at 102,000^. The protein content of the
extracts was then determined by the biuret colorimetric method and adjusted
to 10 mg protein/ml by addition of buffer. This solution could be stored at
0° C for 24 hours before examination, but freezing and thawing caused altera-
tions in ultracentrifuge patterns. When glucose was the source of carbon,
irrespective of whether it was limiting for growth or supplied in excess, or
whether (NH^SOi or peptone was the nitrogen source, cells in mid-logarith-
mic phase gave patterns with boundaries that sedimented at about 40, 29, 20,
and 8 S.

It was only when cell division had ceased for several hours that modifica-
tions could be seen, namely, a reduction in the size of the 40 S peak and the
appearance of a small 13 S peak. By contrast, when cells grown on glucose
were incubated in a lactose growth medium, a 13 S boundary was observed
for actively dividing cells. When lactose was utilized as sole carbon source in
a mineral salts medium there was a lag of 80 minutes before cell division
began, but the (3-galactosidase activity of the culture was increasing in this
period and a trace of the 13 S component appeared in that time. When pep-
tone was the nitrogen source, 3-galactosidase was synthesized much faster and
the lag preceding cell division was only 20 minutes.

Ultracentrifuge patterns for extracts from these cells are shown in figure 1,
where, since photographs were taken at 32 minutes, the 40 S boundaries have
already sedimented. After incubation with lactose for 100 minutes, peaks of
29, 20, and 13 S are well defined. Extracts from cells at this stage of growth
on glucose show only 29 and 20 S boundaries in addition to 8 S after centrifug-
ing for 32 minutes, the trace of 13 S component in the initial stationary phase
culture being lost during cell division. When cells were adapted to utilize galac-
tose and D-xylose as sources of carbon, boundaries that sedimented at about
13 S also appeared; and they were visible in extracts containing the induced
enzyme citratase (Dagley and Sykes [6]).

A correlation may therefore be suggested between the appearance of 13 S
components and the induction of certain enzymes, including those that may
be developed by glucose-grown cells as they remain in the stationary phase in
the presence of accumulated products of metabolism. The significance of such
a correlation, however, is not evident. It is possible, for example, that 13 S

MICROSOMAL PARTICLES

Fig. 1. Patterns at 32 minutes and 187,000^ for extracts from E. coli during growth in
lactose medium. Cells were harvested from: (a) a stationary-phase glucose mineral salts
culture; (b) lactose medium when in early logarithmic phase, 30 minutes after transfer
from (a); (c) 100 minutes after transfer; (d) after 160 minutes. Sedimentation is to the
left, and visible peaks have sedimentation coefficients of 29, 20, 13, and 8 S. In each pattern
the 40 S boundary has already sedimented.

components might have some general significance in enzyme induction proc-
esses, or, alternatively, that the actual enzyme molecules are synthesized in
amounts sufficient to affect the ultracentrifuge patterns and that, by coincidence,
the molecules of those we have studied are all of the size (and shape) to sedi-
ment at about 13 S.

Data tending to favor the second suggestion were obtained by following the
sedimentation of enzyme activities in extracts. For these measurements, the
rotor was allowed to come to rest after a field of 187,000g had been maintained
for a definite time interval, and the cell was carefully removed. By means of
a syringe it was possible to withdraw almost the whole of the supernatant
without disturbing the pellet deposited in the cell. The activity remaining in
the supernatant was then determined, and the sequence of operations was re-
peated for a different length of centrifugation so that a graph could be con-
structed to relate duration of spin to supernatant activity.

The enzymes assayed were arginine, lysine, and glutamate decarboxylases
(Gale [7]) for extracts from cells grown in media containing 2 per cent glucose
and 1 per cent peptone with addition of the corresponding amino acids; cit-
ratase using the media and methods of Dagley and Sykes |6); and the 3-galac-
tosidase (Lederberg [8]) of cells grown at the expense of lactose. The consti-
tutive enzymes of the TCA cycle, malic and isocitric dehydrogenases, fumarase
and aconitase, present in extracts of cells grown in mineral salts media with
limiting glucose, were also assayed by the spectrophotometric methods used by
Englesberg and colleagues [9, 10].

In figure 2 it is seen that centrifuging for about 90 minutes removed all the
citratase, (3-galactosidase, and glutamic decarboxylase of extracts. This is the
time taken to sediment the 13 S boundary. Lysine and arginine decarboxylases
sedimented faster, at about the speed of the 20 S boundary. Malic and isocitric
dehydrogenase activity moved much more slowly, and fumarase and aconitase
appreciably slower than 13 S. The behavior of the four TCA-cycle enzymes
is in agreement with the view that they were present in these extracts as indi-
vidual molecules, since an approximate molecular weight of 40,000 has been

DAG LEY AND SYKES

assigned to malic dehydrogenase (Wolfe and Nielands [11]), of 64,000 to iso-
citric dehydrogenase (Dixon and Moyle [12]), and 204,000 to fumarase (Mas-
sey [13]; Cecil and Ogston [14]). If an estimate of 700,000 is taken as the
molecular weight of (3-galactosidase (Cohn [15]), the enzyme is certainly no
smaller than the 13 S component and its synthesis during adaptation to lactose
might well result in an addition to the pattern in this region. If this is also
true for the other induced enzymes it is surprising that they appear to be syn-
thesized in such quantity and that the molecules are so large that they sediment
between 13 and 20 S.

It is sometimes stated that a number of enzymes are located in the 40 S
component, but this is not so for the activities we have investigated. In this
connection, observations on another system studied in these laboratories are
relevant (Callely, Dagley, and Hodgson [16]). Extracts have been prepared
from a vibrio which catalyze the oxidation of octanoate and other fatty acids
to acetate, apparently by the reactions of the fatty acid spiral (Lynen and
Ochoa [17]). If there are associations of related biochemical activities upon the
40 S particle analogous to those present in various particles from higher organ-
isms, they might well be sought here; but in fact the component may be re-
moved from these extracts with little diminution of the over-all rate of oxidation.

O
D

o
o

0 5

30 60

Time of cenfrifuging, minutes

Fig. 2. Sedimentation of enzymes at 187,000^. Experimental points for: (3-galactosidase,
solid circle; citratase, square; glutamic decarboxylase, open circle. Arrow a shows time
when no lysine or arginine decarboxylase remained; and b, c, d, and e show levels of super-
natant activities after 90 minutes for fumarase, aconitase, isocitric dehydrogenase, and malic
dehydrogenase, respectively.

66 MICROSOMAL PARTICLES

THE 40 S COMPONENT

Variations in the concentration of the 40 S component due to cultural changes
were far greater than for 13 S. Cells grown in peptone contained higher con-
centrations of the 40 S component than those at a corresponding phase of
growth in mineral salts media, and for both types of medium the concentration
increased during cell division and decreased in the stationary phase. Since the
40 S component carries most of the RNA of E. coli (Schachman et al. [1]), these
observations agree with Wade and Morgan [18], who found a higher RNA
content for dividing than for resting cells; but we have never examined extracts
of cells harvested from stationary-phase cultures that were entirely devoid of
40, 29, or 20 S components although we adopted the same extraction procedures
as these authors in several experiments. Cells that had remained 2 hours in a
stationary culture were not distinguishable from those late in logarithmic
growth; and a significant reduction in the 40 S component could only be seen
after 8 hours.

The most striking effect observed was the disappearance of the 40 S boundary
when cells were resuspended in phosphate buffer of the same strength (0.13 M)
as that in the growth medium from which they were harvested. Apparently
no permanent damage is done to the cells by this treatment because, on addi-
tion to the buffer of glucose, Mg++ and (NH4) 2SO4 or peptone, growth occurs
with little or no lag period. These observations are illustrated in figure 3 with
patterns of extracts from cells grown to the stationary phase in mineral salts
medium (a), which were then transferred to peptone medium and examined
in mid-logarithmic phase (b) and at the end of this phase (c). The augmen-
tation of 40 S during cell division in peptone is evident; and, in (c), 29 S has
also increased. Cells that gave pattern (c) were incubated for 30 minutes in
0.13 M phosphate, and most of the 40 S disappeared to leave two small peaks
of 43 and 40 S (d) ; on further incubation only traces of material remained
that sedimented in this region (e). Peptone and glucose were now added to
the suspended cells; growth resumed, and (/) gives the pattern after 1 hour.

Some of the conditions for stabilizing the 40 S component within whole cells
were investigated. Dr. A. Tissieres informed us that its disappearance de-
pended on the concentration of phosphate in the buffer used to suspend the
cells, and we have found a considerable diminution of the rate of loss of 40 S
when the phosphate is reduced from 0.13 M to 0.066 M. It does not appear that
the effect of orthophosphate is specific, however, since disappearance of the
40 S component has been shown for whole cells resuspended in potassium
chloride of the same ionic strength as 0.13 M phosphate. The importance of
magnesium ions is shown by the results of figure 4. If the growth medium is
complete except for addition of Mg++, 40 S is lost (fig. 4£). On addition of
Mg++, 40 S is stabilized and cell division may begin; but Mg++ may stabilize this
component even in cells that are not able to divide because the source of energy
has been omitted (fig. 4<r). Wade and Morgan [18] have shown an association

DAGLEY AND SYKES

(*)

ifiB a I P»l _■ ; I ■ *»*..■ .w,.,^..-.,Jl

(/)

Fig. 3. Patterns after 16 minutes' centrifuging at 187,000g for extracts from cells, in
stationary-phase glucose mineral salts culture (a); in mid-logarithmic phase after transfer
to glucose peptone medium (b) ; in late logarithmic phase in this medium (c) ; after incu-
bation with 0.13 M phosphate buffer, pH 7, (d) for 30 minutes and (<?) for 120 minutes.
The volume of buffer for resuspension was the same as that of the culture from which the
cells were taken. On addition of glucose and peptone to the suspension the cells grew, and
pattern (/) was given after 1 hour.

of magnesium ions with an ultracentrifuge fraction that contains the 40 S
component, and, drawing attention to the observation of Webb [19] that bac-
teria may synthesize protein but may not divide when there is a deficiency of
magnesium, they make the stimulating suggestion that the RNA in this frac-
tion is directly concerned with the cell-division process.

Without further study we cannot conclude that the stability of the 40 S com-
ponent in whole cells is determined solely by the concentrations of Mg++ and
other inorganic ions in the medium. Thus it appears that when cells are grown
in a rich peptone medium they are less readily depleted of 40 S than when
grown in mineral salts; and, in our studies of 3-galactosidase development dur-
ing the lag in cell division in a lactose mineral salts medium, we observed a
reduction in this component although the magnesium content of the culture
was normal. It is therefore possible that other factors concerned with the
metabolism of the cell may also control the stabilization or degradation of the
40 S macromolecule.

When a cell-free extract was diluted, or the buffer concentration used in its

MICROSOMAL PARTICLES

(a) (b) (c)

Fig. 4. Patterns, after 16 minutes' centrifuging at 187,000^, for extracts from cells grown
in a glucose mineral salts medium and then resuspended in solutions containing 1 g
(NH4)2S04 per liter of 0.13 M phosphate buffer, pH 7, with no further addition (a);
addition of 0.01 M glucose (b); addition of 0.2 g MgS04'7H20 per liter (c). Cells were
incubated for 90 minutes in the same volume of solution as that from which they were
harvested.

preparation was increased, the proportion of 40 S relative to the other compo-
nents was reduced. The patterns shown in figure 5 suggest a split of 40 S
macromolecules into 29 and 20 S on dilution, favored by high phosphate con-
centration. Thus, no 40 S boundary was visible in 0.2 M phosphate: the lead-
ing peaks in the extract which contained 7.5 mg protein/ml sedimented at
measured (uncorrected) speeds of 27 and 20 S respectively; and from their in-

1, j H

ILJl

M i

(*)

Fig. 5. Patterns, after 16 minutes' centrifuging at 187,000g-, for extracts prepared in
three strengths of phosphate buffer. Extract concentrations were: 10 mg protein/ml (a);
7.5 mg protein/ml (b). Sedimentation to the left; in 0.066 M and 0.04 M phosphate,
boundaries of 40, 29, 20, and 8 S are visible. The three photographs of either scries were
taken at the same schlieren angle; but an adjustment was made between series to provide
comparable areas for the 8 S peak.

DAGLEY AND SYKES

creased areas it appears that the components forming these boundaries may
contain the material that appears as 40 S at lower phosphate concentrations. In
0.066 M phosphate, the leading boundary sedimented at 40 S followed by 26 S
and 19 S at 10 mg protein/ml, but there was dissociation of the 40 S component
at 7.5 mg protein/ml. In 0.04 M phosphate, 40 S was stable at both concentra-
tions of extract. It is of interest that concentrations of phosphate above 0.06 M,
but not below, effect a loss of the 40 S components whether they are inside the
cells or in extracts isolated from them. This supports the evidence of Roberts
et al. [20] that the phosphate concentration inside E. coli does not differ greatly
from that outside. The final results of disintegration of the 40 S component,
however, are not the same in whole cells as in extracts, for 29 and 20 S com-
ponents appear to be the main disintegration products in extracts, whereas in
whole cells these peaks are not strongly augmented. It is possible that the ini-
tial split of 40 S is to give 29 and 20 S components and that in whole cells the
process goes further to produce lower-molecular-weight diffusible material.

We are grateful to the Medical Research Council for their financial support
of this work.

REFERENCES

1. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochem. Biophys.,
38, 245 (1952).

2. A. Siegel, S. J. Singer, and S. G. Wild-
man, Arch. Biochem. Biophys., 41, 278
(1952).

3. D. Billen and E. Volkin, /. BacterioL,
67, 191 (1954).

4. M. Stephenson, Bacterial Metabolism,
3d ed., p. 311, Longmans, Green and Com-
pany, London, 1949.

5. D. E. Hughes, Brit. J. Exptl. Pathol.,
32, 97 (1951).

6. S. Dagley and J. Sykes, Arch. Bio-
chem. Biophys., 62, 338 (1956).

7. E. F. Gale, Advances in Enzymol., 6,
1 (1946).

8. J. Lederberg, /. BacterioL, 60, 381
(1950).

9. E. Englesberg, J. B. Levy, and A.
Gibor, /. BacterioL, 68, 178 (1954).

10. E. Englesberg and J. B. Levy, /. Bac-
terioL, 69,418 (1955).

11. R. Wolfe and J. Nielands, /. Biol.
Chcm., 221, 61 (1956).

12. M. Dixon and J. Moyle, Biochem. ].,
63, 548 (1956).

13. V. Massev, Biochem. J., 51, 490
(1952).

14. R. Cecil and A. G. Ogston, Biochem.
J., 51, 494 (1952).

15. M. Cohn, BacterioL Revs., 21, 140
(1957).

16. A. G. Callely, S. Dagley, and B.
Hodgson, Biochem. J., 66, 47P (1957).

17. F. Lynen and S. Ochoa, Biochim. et
Biophys. Acta, 12, 299 (1953).

18. H. E. Wade and D. M. Morgan,
Biochem. /., 65, 321 (1957).

19. M. Webb, /. Gen. Microbiol., 3, 410
(1949).

20. R. B. Roberts, P. H. Abelson, D. B.
Covvie, E. T. Bolton, and R. J. Britten,
Studies of Biosynthesis in Escherichia coli,
Carnegie Inst. Wash. Publ. 607, Washing-
ton, D. C, 1955.

Physicochemical and Metabolic Studies on
Rat Liver Ribonucleoprotein1

MARY L. PETERMANN MARY G. HAMILTON

M. EARL BALIS KUMUD SAMARTH - PAULINE PECORA

Sloan-Kettering Institute for Cancer Research
and Sloan-Kettering Division, Cornell University Medical College

The presence in uninfected tissues of particles with sedimentation constants
of about 75 S has long been known to virologists [1]. Similar macromolecules
have been found in tumors (Kahler and Bryan [2]). We first observed them
in spleen [3], and later in liver, pancreas, and various tumors [4]. They are
now known to be ribonucleoproteins (RNP) [5]. Ultracentrifugal patterns of
these RNPs show a number of boundaries. Their sedimentation coefficients
are so strongly dependent on concentration that we have denoted them by
letters, such as B, C, and E, rather than identifying them by their sedimenta-
tion constants at infinite dilution. B has a sedimentation constant of 78 S, C of
about 62 S, and E of 46 S, corresponding roughly with the S80, S60, and S40
boundaries found in nucleoproteins from microorganisms.

In general, where we find a large amount of nucleoprotein B, as in pancreas
and normal liver [4], the electron microscopists find most of the granules at-
tached to endoplasmic reticulum [6] ; but where we find increased amounts of
C and E, as in liver tumors [4], the electron microscopists find granules not
attached to reticulum [7]. We therefore wanted to see whether a large micro-
some fraction, containing only RNP bound to endoplasmic reticulum, con-
tained only B, while a small microsome or ultramicrosome fraction containing
free RNP particles was rich in C and E.

1 The authors wish to acknowledge the assistance of the Atomic Energy Commission
(Contract no. AT (30-1)— 910), and the National Cancer Institute of the United States
Public Health Service (grants nos. C-2329 and CY-3190).

2 Visiting research fellow.

PETERMANN ET AL.

We prepared such fractions by differential centrifugation at 78,000g in 0.79
M sucrose. The whole microsome fraction was centrifuged for 5 hours. The
large-microsome fraction was centrifuged for only 50 minutes, in order to sedi-
ment only large fragments of endoplasmic reticulum. From this supernatant
the small-microsome fraction was sedimented for 5 hours. Each fraction was
washed by resedimentation in 0.79 M sucrose containing 5 X 10~4 M K2HPO4
and KH2PO4, and 5 X 10"4 M MgCl2 to preserve the RNP [8], and finally was
suspended in this same buffer without the sucrose, or in water, to a RNP con-
centration of about 5 mg/ml. For ultracentrifugal analysis each sample was
diluted with 0.2 volume of a fivefold concentrated buffer. Two sets of analyses
carried out in 0.1 M KHCOs are shown in figure 1. When a mere trace of
magnesium is present, as in the upper row, the RNP from the large micro-
somes is chiefly B, with only small amounts of C and E. The small microsomes,
however, do show considerable amounts of C and E. With 0.0024 M magne-
sium, in the bottom row, all preparations show less C and E, but the same rela-
tionship is retained — there is more C and E in the small microsomes than in
the large ones. The whole microsome fraction falls in between, as one would
expect. Varying the pH or the concentrations of monobasic and dibasic ions
gives an assortment of patterns, but there is always more C and E in the ex-
tracts of the small microsomes.

Whether any of the RNP in the small-microsome fraction is really "free" in
the liver cell, however, is difficult to determine. In extracts made in water or
the dilute phosphate-magnesium buffer, at pH 7, no RNP boundaries are seen
in the ultracentrifuge. They appear when the pH is raised to 8.0 or the ionic
strength is increased. Since the state of the particles is so dependent on their
ionic environment, they will have to be studied in buffers which approximate

FRACTION
WHOLE LARGE

BCE

SMALL

BC E

♦ i ♦

k^/_jLV ^J^J

SOLVENT

pH KHCO3 Phos. MgCI2
M M M

8.0 O.IO

tr.

■i

8.0 O.IO 0.0008 0.0024

Fig. 1. Ultracentrifugal patterns of RNP extracted from microsomal fractions. The
pictures were taken after 14 minutes at 37,020 rpm.

72 MICROSOMAL PARTICLES

AFTER 3 WASHES AFTER BARIUM PRECIPITATION

Fig. 2. Electrophoretic patterns of purified RNP in 0.10 M KHC03, pH 8.2, contain-
ing 0.001 M MgCl2. Pictures taken after 60 minutes at 5.2 volts/cm. The left-hand pat-
terns show RNP that had been washed three times. The right-hand patterns show the
same material after removal of the ferritin. (See text.)

the ionic composition of the liver cell before any conclusions can be drawn.3
Most of our recent work has been carried out on purified RNP [9]. The
microsomes are disrupted with deoxycholate, and the RNP is purified by alter-
nate cycles of high- and low-speed centrifugation. Recently we have made some
improvements in the procedure. The livers do not have to be perfused. The
addition of penicillin (100 units/ml) to all the solutions increases the stability.4
Instead of washing with calf-liver dialysate we now use the potassium phos-
phate-magnesium chloride buffer; its pW is about 7. The washed RNP still
shows about 5 per cent of ferritin on electrophoretic analysis (fig. 2). This can
be removed by precipitating the RNP with 0.005 M barium acetate and redis-
solving it by dialysis against 5 X 10"4 M K2HPO4, KH2P04, and MgS04 (fig. 2) .
We have carried out extensive studies of the stability of purified RNP. Like
the crude microsomal extracts, the purified RNP is very sensitive to pH, ionic
strength, and dibasic and monobasic cations. Figure 3 shows the effect of mag-
nesium in the presence of 0.1 M NaHCOs. Fresh RNP looks like the third

3 Recently we have prepared large- and small-microsome fractions by sedimentation in
0.20 M sucrose, and resuspended them in water at pH 7.1 without washing. On ultracen-
trifugal analysis sizable C and E boundaries were observed in the small-microsome frac-
tion, although none were apparent in the large-microsome fraction.

4 The purification of the RNP requires about 3 days; although the preparation is kept
cold, unless penicillin or sterile technique is used the RNP has an odor like that of spoiled
meat. This may be due to cadaverine produced from N-terminal lysine by bacterial lysine
decarboxylase; after treatment of the RNP with dinitrofluorobenzene and acid hydrolysis,
the only DNP amino acid detectable is lysine.

PETERMANN ET AL. 73

picture down, chiefly B. After dialysis overnight against bicarbonate contain-
ing 0.005 M MgCl2 the RNP is unchanged. With less magnesium, as shown
in the two upper pictures, it dissociates; with more magnesium, as shown in
the bottom row, the B boundary gets smaller, because the RNP has begun to
aggregate and precipitate out.

In the purified RNP we found 40 per cent RNA on a dry-weight basis [9].
Similar values have been found for purified RNPs from yeast [10] and pea
seedlings [11], again on a dry-weight basis. When RNA is compared with
protein nitrogen, on the other hand, we and many other workers find values
for RNA of 50 to 60 per cent. Some of this discrepancy is probably caused by
lipid, which contributes more to the dry weight than to the nitrogen. Some of
it, however, seems to occur because hot 5 per cent trichloroacetic acid extracts
some other nitrogen besides that in the RNA. With 10, 15, and 20 per cent
trichloroacetic acid we still find extra nitrogen in the extracts.

A B E S PH NaHC03 Phos. MgCI2

+ + + + M MM'

82 O.IO O.OOI

8.2 O.IO O.OOI 0.0005

8.2 0.10 0.001 0.0050

8.2 0.10 0.001 0.0100

Fig. 3. The effect of magnesium on the stability of purified RNP. Ultracentrifugal
patterns were obtained after 14 minutes at 37,020 rpm.

We have carried out a number of metabolic studies on purified RNP con-
taining about 4 per cent ferritin. Figure 4 shows the incorporation of glycine-
1-C14 into adenine and peptide glycine. The upper row shows the time course
of the incorporation in normal male rats. Fifteen minutes after the injection
of the isotope, by tail vein, the specific activity of the acid-soluble adenine of
the liver was quite high, whereas that of the adenine in the purified RNP was
still very low, and, although it increased very slowly with time, it was still low
at 17 hours. The specific activity of the liver nucleoprotein glycine was highest

MICROSOMAL PARTICLES

_j
o

o
rr
o

I-
=>

200
0

NORMAL

I J -W

GROWTH HORMONE

•Hh

1200

800

• ACID SOLUBLE ADENINE
■ ADENIf
B GLYCINE

iine]

NE J

0 GLYCINE-SUPERNATANT

PROTEIN
Q GLYCINE-SERUM PROTEIN

til

JK&l

V4 i

TIME IN HOURS

o
400 o

0 ffi

Q.
Ul

2000 z>

1600 cr
ui

1200

800

400

2
O

I
LiJ

O
?J

The incorporation of glycine-1-C14 into the adenine and glycine of rat liver RNP,
and into "supernatant" and serum proteins (see text).

Fig. 4.

at 15 minutes and then dropped off. The glycine of the liver supernatant pro-
teins, plus the microsomal lipoproteins soluble in deoxycholate, reached a maxi-
mum between 1 and 2 hours, and then fell. The glycine of the serum proteins
increased very slowly, and was highest at 17 hours. Similar results have been
obtained with labeled adenine and methionine as tracers.

The lower row shows the results obtained on rats pretreated with bovine
growth hormone, 410 [\g per rat per day, for 2 weeks. There was no effect on
the RNP adenine, but the effects on the three protein fractions were marked.
Everything seemed to have been speeded up. The nucleoprotein glycine ac-
tivity dropped more rapidly; the liver supernatant activity rose more steeply;
and the serum proteins apparently reached a maximum at some time before 17
hours, and then declined again.

These results illustrate, first, the lack of correspondence in the extents of
isotope incorporation into the RNA and the total protein glycine of the RNP,
and, second, the effect of growth hormone, which speeded up the incorporation
of glycine into protein without any change in the rate at which the particle
RNA became labeled. From these observations we conclude that, at least in
rat liver, the total RNA of the RNP cannot be functioning as an active template.

PETERMANN ET AL.

REFERENCES

1. A. R. Taylor, D. G. Sharp, and B.
Woodhall, Science, 97, 226 (1943).

2. H. Kahler and W. R. Bryan, /. Natl.
Cancer Inst., 4, 37 (1943-1944).

3. M. L. Petermann and M. G. Hamil-
ton, Cancer Research, 12, 373 (1952).

4. M. L. Petermann, N. A. Mizen, and
M. G. Hamilton, Cancer Research, 16, 620
(1956).

5. M. L. Petermann, M. G. Hamilton,
and N. A. Mizen, Cancer Research, 14,
360 (1954).

6. G. E. Palade, /. Biophys. Biochem.
Cytol, 1, 59 (1955).

7. A. F. Howatson and A. W. Ham,
Cancer Research, 15, 62 (1955).

8. F.-C. Chao, Arch. Biochem. Biophys.,
70, 426 (1957).

9. M. L. Petermann and M. G. Hamil-
ton, /. Biol. Chem., 224, 725 (1957).

10. F.-C. Chao and H. K. Schachman,
Arch. Biochem. Biophys., 61, 220 (1956).

11. P. O. P. T'so, J. Bonner, and J. Vino-
grad, /. Biophys. Biochem. Cytol., 2, 451
(1956).

Ultracentrifugal Studies of Microsomes from

Starving, Nonproliferating, and

Proliferating Yeast

JAMES K. ASHIKAWA

Dontier Laboratory of Biophysics and Medical Physics
University of California, Berkeley

In the decade since Claude's successful isolation of microsomes from liver
and other tissue homogenates by differential centrifugation [1, 2], considerable
progress has been made toward elucidating their biochemical and morphological
characteristics. The microsomal "ribonucleoprotein" particles isolated from bac-
terial cells and from plant and animal tissues are rich in ribonucleic acid [3-10].
In electron-microscopic observations these microsomal particles, either isolated or
in intact plant and animal cells, appear as spherical particles with diameters
ranging from 100 to 400 A. They occur either bound to the endoplasmic reticu-
lum or freely dispersed in the cytoplasmic matrix [7-14].

There are interesting studies indicating that microsomes may be actively in-
volved in protein and lipid synthesis [5, 15, 16]. In vivo [17-20] and in vitro
studies [15, 20, 21] have shown that labeled amino acids are preferentially in-
corporated into the microsomal proteins, suggesting synthetic activity.

Since these microsome particles appear to be functional organelles in both
plant and animal cells, it is highly probable that their physicochemical proper-
ties will be altered by varying the physiological state of the cell. Preliminary
studies of microsomes isolated from starving, nonproliferating, and proliferat-
ing yeast cells have shown that several new ribonucleoprotein particles appear
during cell division [22, 23]. This paper presents further evidence thereon.

EXPERIMENTAL PROCEDURE AND RESULTS

Haploid yeast cells (Saccharomyces cerevisiae strain S. C. 7) aerobically cul-
tured for 24 hours at 30° C in yeast extract and dextrose ( YED 1 : 2 per cent)

ASHIKAWA

were harvested and washed twice with sterile distilled water. Aliquots of cells
were then suspended in nitrogen-deficient medium (4 per cent dextrose : M/GO
KH2PO4:3/60 M NaHsPOi) and aerated for 48 to 11 hours at 30° C. During
this nitrogen starvation, the buffered dextrose medium in some of the cultures
was changed every 12 hours. At the end of starvation, the cells were centrifuged
and rewashed twice with sterile distilled water. Approximately 6 gram aliquots
of wet cells were suspended in 2-liter flasks containing 1500 ml YED (1:2 per
cent) and induced to grow aerobically at 30° for varying time intervals. The
growth curve of the cells and the corresponding bud counts for the first 26
hours are shown in figure 1. The yeast cells were then harvested at different
times, and microsomal particles were isolated according to Wolfe [23]. Cells
hand-ground with 100-grid Carborundum in mortar and pestle at 0° C were
extracted with several volumes of solvent (0.00125 M KH2PO4-K2HPO4 3:7,

lOOrr

_l
_l
UJ

r-
O

"i r

-o-

8 12 16 20 24 28

HOURS AFTER INOCULATION
Fig. 1. Relationship of budding to growth of yeast cells, (a) Growth curve of 48-hour
nitrogen-starved yeast cells aerobically cultured at 30° C in YED (1:2 per cent), (b) Curve
correlating percentage of visible buds to corresponding growth stages.

MICROSOMAL PARTICLES

0.001 M MgCl2, and 0.01 M KC1). After vacuum nitration through celite filter-
aid, the microsomes were sedimented from the filtrate by ultracentrifugation in
the preparative ultracentrifuge (Spinco Model L) at 114,000g (40,000 rpm) for
1 hour. The translucent microsomal pellets isolated from yeast cells in different
growth stages were carefully redissolved in the buffered solvent and analyzed
in the analytical ultracentrifuge (Spinco Model E) at 200,000^. Figure 2 shows

(«)

(c)

Fig. 2. Sedimentation photographs of microsome particles from yeast-cell extracts
6 minutes after up to speed (UTS) at 200,000,? in the analytical ultracentrifuge (Spinco
Model E). (a) Top: Cell extract from 48-hour nitrogen-starved cells corresponding to the
inoculum cells at time 0. Bottom: Extract from nitrogen-starved cells 1.5 hours after cells
given utilizable nitrogen, (b) Top: After 3 hours. Bottom: After 5 hours, (c) Top:
10 X concentrated extract from nitrogen-starved cells 7 hours after cells given utilizable
nitrogen. Bottom: Extract from nitrogen-starved cells 10 hours after cells given utilizable
nitrogen, (el) Top: After 26 hours. Bottom: After 52 hours.

ASH IK AW A

the changes in the sedimentation pattern which indicate the appearance of new
microsomal particles [22, 23]. During the lag phase when less than 3 per cent
of the cells have visible buds, only the 80 S component (a peak in fig. 3) is pres-
ent in the ultracentrifuge pattern of cytoplasmic extract. As the cells begin to
proliferate and enter the log phase of growth (40 to 50 per cent visible buds),
four new microsomal components (3, y, 5, and £ peaks in fig. 3) appear in the
cell extracts. From log growth phase through stationary phase, the only change
in the sedimentation pattern appears to be in the ratio of these components.
Cell starvation is first evidenced by a decrease in the stability of the micro-
somes, which is indicated by the presence of polymerized material that sedi-

(«)

(*)

(/)

Fig. 3. Sedimentation photographs of microsomes isolated from 48-hour stationary-phase
cells 6 minutes after UTS at 200,0(%. (a) Yeast extract suspended in phosphate buffer
(0.00125 M KH2P04-K2HP04 3:7, 0.001 M MgCU, and 0.01 M KCl) at 4° C for 1 day. (b)
Yeast extract suspended" in phosphate buffer at 4° C for 2 days, (c) After 7 days, (d)
After 10 days. (<?) After 13 days. (/) After 27 days.

MICROSOMAL PARTICLES

(«)

(c)

Fig. 4. Sedimentation photographs of isolated 80 S particles taken at 0, 2, and 4 minutes
after UTS at 200,0C% in the analytical ultracentrifuge. Particles moving from left to
right, (a) 80 S particles from 24-hour stationary-phase cells observed immediately after
isolation, (b) 80 S particles, isolated from 24-hour stationary cells, suspended in phosphate
buffer and kept at room temperature for 2 days, (c) 80 S particles isolated from cells
nitrogen-starved for 72 hours suspended in phosphate buffer and kept at room temperature
for 2 days.

ASH IK AW A 81

ments as a rapidly spreading fore peak [23] before the centrifuge is up to speed
(UTS). The growing cell components disappear as starvation progresses.

The stability of microsome components isolated from log phase, stationary
phase, and starving cells and kept at 4° C or at room temperature in buffered
solvent was also studied. There was no detectable difference in stability between
particles isolated from log phase and those isolated from stationary phase.
Figure 3 shows the degradation of microsomes isolated from 48-hour stationary-
phase cells and suspended in phosphate buffer at 4° C.

Since only the 80 S component is present in old stationary-phase cells and in
starving cells, the stability of this component isolated from proliferating, non-
proliferating, and starving cells, and dissolved in buffered solvent, was com-
pared. Figure ^a shows the sedimentation pattern at 0, 2, and 4 minutes after
UTS at 200,00% of the 80 S particles isolated from 24-hour stationary-phase
cells. The sedimentation pattern of this component from 72-hour nitrogen-
starved cells is similar, except for the broad, rapidly spreading, and sedimenting
fore peak. As shown in figure %, the 80 S component isolated from log-phase
and stationary-phase cells gave rise to the 60 S and 40 S component [24] when
kept at room temperature for 2 days. On the other hand, the 80 S component
isolated from cells nitrogen-starved for 72 hours became degraded by forming
rapidly sedimenting aggregates. See figures 4c and 5.

DISCUSSION

When starved cells are given utilizable nitrogen, the growth curve shows a
characteristic lag phase corresponding to the degree of starvation. During this
phase the 80 S component appears to be degraded and reconstituted. As shown
in figure 2, a decrease in the 80 S component is noted 1.5 hours after starved
cells are given utilizable nitrogen. Two hours after inoculation, the microsomal
concentration appears approximately equal to that of the inoculum cells. At 3
hours, new microsomal components appear in the cytoplasmic extract. The cells
are now entering the log growth phase, during which the microsome concen-
tration reaches a maximum. This condition is followed by a gradual quantita-
tive change in the microsomal components as the cells pass through the sta-
tionary phase.

Chao and Schachman [8, 24] have shown that, in vitro, altering the ionic
environment of the solvent will dissociate or aggregate the 80 S component. It
would therefore be interesting to ascertain whether a similar mechanism is re-
sponsible for changes in the microsomal components in respiring cells.

Since the 80 S particles from only starving cells of low viability are degraded
by forming rapidly sedimenting aggregates (figures 4c and 5), there appears
to be a correlation between call viability and the chemical state of this particle.

Furthermore, if microsomes are involved in the synthesis of proteins and lipids
[5, 15, 16], the changes observed in the microsomal components with cell growth
could be a mechanism that controls their synthetic activities.

MICROSOMAL PARTICLES

Fig. 5. Sedimentation photographs at UTS (200,0(%) of 80 S particles isolated from
cells nitrogen-starved for 72 hours and suspended in phosphate buffer at 4° C. (a) After
1 day. (b) After 2 days, (c) After 7 days, (d) After 11 days.

SUMMARY

Ultracentrifugal studies of yeast-cell extracts have shown a correlation be-
tween the physiological states of the cells and the stability and appearance or
disappearance of microsomal particles from the cytoplasm.

ACKNOWLEDGMENT

This work was done in the Biophysics group of Donner Laboratory and was
supported by contract with the Atomic Energy Commission. I wish to thank
Dr. Cornelius A. Tobias for his interest in this work.

ASHIKAWA

REFERENCES

1. A. Claude, Science, 97, 451 (1943).

2. A. Claude, Harvey Lectures, 48, 121
(1947-1948).

3. C. P. Barnum and R. A. Huseby,
Arch. Biochem., 19, 17 (1948).

4. G. E. Palade and P. Siekevitz, Fed-
eration Proc, 14, 262 (1955).

5. J. W. Littlefield, E. B. Keller, J. Gross,
and P. C. Zamecnik, /. Biol. Chew., 217 ,
111 (1955).

6. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochem. Biophys., 38,
245 (1952).

7. P. O. P. Ts'o, J. Bonner, and J. Vino-
grad, /. Biophys. Biochem. Cytol., 2, 451
(1956).

8. F. Chao and H. K. Schachman, Arch.
Biochem. Biophys., 61, 220 (1956).

9. G. E. Palade and P. Siekevitz, /. Bio-
phys. Biochem. Cytol. , 2, 171 (1956).

10. G. E. Palade and P. Siekevitz, /. Bio-
phys. Biochem. Cytol, 2, 671 (1956).

11. G. E. Palade, /. Biophys. Biochem.
Cytol, 1, 59 (1955).

12. G. E. Palade and K. R. Porter, /. Ex-
ptl.Med., 100, 641 (1954).

13. K. R. Porter, /. Exptl. Med., 97, 727
(1953).

14. D. B. Slautterback, Exptl Cell Re-
search, 5, 173 (1953).

15. P. Siekevitz, /. Biol. Chem., 195,
549 (1952).

16. H. P. Klein, /. Bacteriol, 73, 530
(1957).

17. T. Hultin, Exptl Cell Research, 1,
376, 599 (1950).

18. N. D. Lee, J. T. Anderson, R. Miller,
and R. H. Williams, /. Biol. Chem., 192,
733 (1951).

19. R. M. S. Smellie, W. M. Mclndoe,
and J. N. Davidson, Biochim. et Biophys.
Acta, 11, 559 (1953).

20. V. Allfrey, M. N. Daly, and A. E.
Mirsky, /. Gen. Physiol, 37, 157 (1953).

21. P. C. Zamecnik and E. B. Keller,
/. Biol Chem., 209, 337 (1954).

22. R. G. Wolfe, UCRL-2553, April
1954.

23. R. G. Wolfe, Arch. Biochem. Bio-
phys., 63, 100 (1956).

24. F. Chao, Arch. Biochem. Biophys.,
70, 426 (1957).

Fractionation of Escherichia coli for
Kinetic Studies

RICHARD B. ROBERTS ROY J. BRITTEN ELLIS T. BOLTON

Department of Terrestrial Magnetism
Carnegie Institution of Washington

A single cell of the bacterium Escherichia coli contains roughly 10,000 ribo-
somes (microsomal particles). If the cells are broken open and their contents
are examined, the analytical centrifuge shows a series of peaks with sedimenta-
tion constants of roughly 20, 30, 40, 60, and 80 S [1-8]. The existence of these
particles in such variety and in such large numbers immediately provokes a
number of questions. Is the rapid growth rate of E. coli a consequence of the
high proportion (25 per cent) of the cellular material that is organized into ribo-
somes? In other words, are ribosomes the sites of protein synthesis in E. coli?
If so, what size of particle is active in protein synthesis ? How are the particles
themselves synthesized? Do the different sizes of particles represent different
stages in the growth of a particle ?

Eventually answers will be found for these questions, but not easily. It will
be necessary to know the composition of the particles— the composition of the
individual classes of particles, not just the composition of a pellet containing an
assortment of particles plus other material. Also the kinetics of isotope incor-
poration will have to be studied. It will not be sufficient simply to deal with
the microsomal fraction, the 100,000g- pellet; rather, the individual groups of
particles will have to be sorted out and measured. Suppose, for example, that
one size of particle is the precursor of another. Kinetic measurements will show
this clearly if the two groups can be resolved; kinetic measurements of both
groups lumped together in a pellet will show nothing.

Chemical fractionation of E. coli gives good separation between the different
classes of compounds, and it is easy to show by kinetic measurements of the
incorporation of radioactive compounds that the small molecules serve as

ROBERTS, BRITTEN, AND BOLTON

precursors of the large ones. In contrast, the simple separation into cell wall,
microsome, and soluble fractions is not sufficient to reveal clearly any precursors
or products among the macromolecules. A further fractionation of the micro-
some pellet is required.

Pellets of somewhat greater homogeneity can be obtained by choosing an
appropriate centrifuging schedule. The material that sediments in 15 minutes at
100,000g- is richer in the large particles than the pellet obtained by centrifuging
down (2 hours at 100,000^) material which stayed in suspension during three
successive 15-minute periods at 100,000^. The composition of the pellet also
varies; the early pellet contains nearly twice the lipid and protein per unit of
nucleic acid. This approach, however, shows no promise of giving adequate
fractionation.

A somewhat better fractionation can be obtained by using the swinging
bucket head for the Spinco Model L centrifuge. Microsome pellets are resus-
pended and layered on top of a sucrose gradient. After a period of centrifuga-
tion, layers are taken off with a pipet. This technique is adequate to show
marked differences in the distributions, depending on the initial material. Fig-
ure 1 shows one curve for a resuspended pellet composed mostly of large (80 S)
particles; another for the smaller particles (20 to 40 S) that result if magnesium
is lacking [8]; and a third, for the nonsedimenting material. The analytical

7 9

Fraction no

Fig. 1. Fractionation of particle preparations using the swinging bucket centrifuge.
Five-tenths milliliter of suspension is placed on top of 4.5 ml sucrose gradient in the cen-
trifuge tube. After 45 minutes at 100,000^, 0.3-ml fractions are taken off from the top with
a pipet.

MICROSOMAL PARTICLES

centrifuge shows that the bottom layers are rich in the heavy particles and lack
the light particles, whereas the top layers show the opposite distribution.

Quite a different type of fractionation results from chromatography on col-
umns of diethylaminoethyl cellulose (DEAE) [9, 10]. Extremely high reso-
lution can be achieved giving a separation of various proteins as shown in
figure 2. Nucleoprotein appears as a prominent peak in the elution diagram
of the total cell juice but not in the diagram obtained with the 100,000^ super-
natant fluid (fig. 3). The corresponding ultraviolet diagrams show that there
are in fact two nucleoprotein peaks: the first peak consists of nucleoprotein of
high molecular weight which can be spun down in the centrifuge; the second

40 60 80 100

Fraction number

Fig. 2. Cell suspension washed and broken as described by Bolton et al. [8] ; 0.5 g wet
weight of cell juice adsorbed on DEAE column (1 cm2 X 20 cm) and eluted with con-
centration gradient 0 to 0.7 M of NaCl in tris-succinate buffer plus magnesium. Lower
curve, total protein indicated by Folin reaction; upper curve, assay for activity of three
different enzymes. One-milliliter samples collected in fraction collector.

ROBERTS, BRITTEN, AND BOLTON

^Totol C«M eitroct

AaJVw^>?V

\ .,100,000 g SN

100
Eluting fluid, ml

150

Fig. 3. Elution patterns of total cell juice and supernatant fluid of 100,000^ 2-hour spin.
Upper curve, optical density at 254, indicating nucleic acid concentration; lower curve,
S35 radioactivity, indicating protein. Note nucleoprotein peak which is missing in 100,00%

SN.

is partly nucleoprotein and partly due to free DNA and RNA which still re-
main in the 100,000^ supernatant fluid.

The elution pattern is not sensitive to the size of the particles. The same pat-
tern is obtained whether the microsome pellet is composed mostly of the large
(80 S) particles or of the smaller 20 to 40 S particles that result from magne-
sium deficiency. Compare figures 4# and b.

Microsome pellets when resuspended and analyzed on the column show the
nucleoprotein peak together with a quantity of other protein which depends on
the method of preparation (fig. 4). A part of the contamination of the micro-
some pellet is due to small bits of cell wall, and another part is due to nonpar-
ticulate protein. In 2 hours at 100,000^ roughly 70 per cent of the 3-galac-
tosidase is sedimented. See also Dagley and Sykes [5, 11]. Accordingly the
least-contaminated preparations of ribosomes are those obtained by resuspend-
ing a microsome pellet and centrifuging again in the swinging bucket head
(fig. 4c).

Unfortunately the column cannot be used to prepare purified ribosomes be-
cause the material eluted from the column is quite different from that origi-
nally adsorbed. When the fractions containing the nucleoprotein peak are

MICROSOMAL PARTICLES

2 I-

a.
O I

TS Microsome pellet

L_ i ^r .^r- -. aa-g t =i

c
■o

"5
o

o
</>

O
IO

TSM

Microsome

pellet j

- i (b)

_. / /
• /

uL.

"7T

1 1 1

i .i/i

1 l l T^f^^O,-^-!

TSM Microsome pellet

3 5 7 9 II 13 15 17 19 21 23 25 27 29 31 33 35
Column fraction no.

Fig. 4. Elution patterns of microsome pellets, a, 100,0C% 2-hour pellet without mag-
nesium present; b, same with magnesium present; c, microsome pellet resuspended and
fractionated with swinging bucket centrifuge.

ROBERTS, BRITTEN, AND BOLTON

centrifuged (100,000^, 2 hours), a colorless glassy pellet is formed which con-
tains approximately 65 per cent of the protein and nucleic acid. This pellet
resuspends easily and completely. The analytical centrifuge shows that it con-
tains peaks in the 20 to 40 S region, whereas the 80 S peak was most promi-
nent in the original material. The ratio of nucleic acid to protein in this pellet
(measured by optical density at 260 mu and S35) is twice that of the starting
material, and the elution pattern obtained when the pellet is rerun on a DEAE
column is very different (fig. 5).

These changes appear to be caused by the column material and not by the
salt of the eluting fluid. Ribosomes exposed to molar NaCl show a reduction
in size but no change in composition or elution pattern.

The fractionation and analysis procedures outlined above are beginning to
yield some useful information about the composition and function of the ribo-
somes. The purified ribosomes are markedly different from the microsome
pellet. For example, the microsome pellet contains considerable phospholipid
(table 1); the ribosomes, little if any. Moreover, the nucleic acid to protein ratio
is somewhat variable in the crude microsome pellet, but the purified ribosomes

S-\~

■o

"l \

— T \ 1 \

/ \ i .\-

"V 1 \

T \»J \

i , -\-

— N-P Peak

-,' ; \

1 , ^B

* 1

1 ' ,,JT \

» •'

' "*Sa ■' \ -V

' * *"\

•I

J "\^

' »\-

! "*~ ^Sw*"

r n ^"^^T

-r-f—f-i i-h-4— +-=m--i i "I i — i— -i —

16 20 24

Column fraction no.

28 32 36 40

Fig. 5.
change to

Nucleoprotein peak of elution pattern spun down and rechromatographed. Note
elution pattern like that of nucleic acid.

90 MICROSOMAL PARTICLES

TABLE 1. Chemical Fractionation of coli Components

Cell Wall

Microsome

Soluble

Whole Cell

30,000^ pellet

100,0(% pellet

100,00% SN

Small molecules

Lipids

RNA

DNA

Protein

Total 100 20 30 50

obtained from the swinging bucket give a constant ratio indicating two amino
acids per nucleotide (NA/P = 60/40 measured by absorption at 260 mu and
Folin [12] test for protein).

The protein of ribosomes differs from other proteins of the cell. Purified
ribosomes were obtained from cells grown with C14 glucose as the sole carbon
source. The protein, after hydrolysis and chromatography, showed an amino
acid distribution in which glutamic acid, alanine, glycine, and lysine were pro-
portionately higher than in the whole cell, whereas methionine and aspartic acid
were lower. In this protein neither cysteine nor cystine seems to be present.

The absence of cystine can best be shown by growing the cells in the presence
of S35Oi to label cystine and methionine. After hydrolysis and chromatography
the radioactivity of methionine and cystine can be measured. In the protein of
the whole cell there is approximately twice as much methionine as cystine [13].
In ribosomes purified in the swinging bucket centrifuge this ratio is 10:1. In
nucleoprotein eluted from the column and sedimented the ratio is greater than
100:1.

Alternatively the lack of cystine can be demonstrated without hydrolysis and
chromatography. Cells containing S35 cystine and S32 methionine were grown
by adding S35Oi and S32 methionine to the medium. To prevent even a slight
leakage of S35 into methionine, a methionine-requiring mutant was used [13].
The sulfur radioactivity per unit protein of the nucleoprotein (obtained by col-
umn analysis of a microsome pellet and sedimentation of the nucleoprotein frac-
tion of the eluate) was 50 times lower than that of the whole cell. Since the
usual occurrence of cystine is only 1 per 60 residues, its occurrence in the nucleo-
protein is less than 1 per 3000.

Kinetic studies of the fractions obtained from the column are also in progress.
S35 has been used to follow incorporation into protein. Exponentially growing
cells were exposed to the tracer for varying periods of time and then broken
and their constituents analyzed. The specific radioactivity of the protein frac-
tions was measured by TCA-precipitable S35 and Folin reaction color. When the
cells are exposed to the tracer for a prolonged period (steady state) the specific
radioactivity varies throughout the elution pattern by a factor of roughly 3,
being lowest in the nucleoprotein fraction. These variations are simply due to
variations in the sulfur content. Other cells were grown for three generations in

ROBERTS, BRITTEN, AND BOLTON

a nonradioactive medium after exposure to the tracer. In this treatment any
intermediates which have a rapid turnover should lose their radioactivity. The
resulting "persistent pattern" was entirely similar to the "steady-state pattern,"
and no protein components could be identified as intermediates.

Finally, cells were exposed to the tracer for short periods. After a 4-minute
exposure the resulting "pulse pattern" was similar to the "steady-state pattern"
except that the radioactivity of the nucleoprotein peak was only one-half of that
expected from the "steady-state" pattern. A similar result was obtained with
cells exposed for 4 minutes to a mixture of C14-labeled amino acids.

Similar experiments carried out with P32Oi give much more striking results.
Figure 6 shows the macromolecular region of the elution patterns obtained with
cells exposed to the tracer for increasing periods of time. The radioactivity ap-
pears first in a quite distinct fraction of the elution pattern, passing through at
a later time to the other regions. In the steady-state and persistent patterns the
phosphorus radioactivity was proportional to the optical density (at 260 mu).
Thus the DEAE column is capable of resolving the nucleic acid and nucleo-
protein into fractions that seem to be precursors and products. Similar kinetic
differences were also observed by Creaser, who used ECTEOLA columns [9]
to analyze alcohol-extracted nucleic acid [14].

The analysis of these data runs into a number of complications. The leading
peak is composed solely of RNP, but the secondary peak is an unresolved mix-
ture of RNP, RNA, and DNA. Furthermore, the pool of low-molecular-weight
precursors to RNA is large and may or may not be in equilibrium with the
smaller pool of DNA precursors [14].

A rough analysis can be made on the basis of several simplifying assump-
tions. Assume first that the low-molecular-weight precursors of the macromole-

DE AE COLUMN ANALYSIS OF E.COLI AT EARLY TIMES AFTER ADDITION OF P32

I 1/2 mm

6min

24 min

Fraction number

Fig. 6. Elution patterns of cell extracts after growing cells were exposed to P3204 for
times indicated. Only a small region of the elution pattern is shown.

MICROSOMAL PARTICLES

cules have the average specific radioactivity of the TCA-soluble pool. Second,
since the persistent and steady-state runs show all the macromolecular ultra-
violet-absorbing material to be uniformly labeled, assume that it is the end
product and that its specific radioactivity is that of the nucleoprotein. Analyzed
on this basis the data point to an intermediate containing roughly 10 per cent
of the nucleic acid.

Some other characteristics of this intermediate have been determined. Very
short exposures to the tracer were used to prepare cell juices which were shown
by column analysis to have most of the P32 in the intermediate and little in the
end products. Most of the low-molecular-weight materials were removed by
washing the cells with water before breaking. This material was analyzed in
the swinging bucket centrifuge. The results (fig. 7) show that the TCA-pre-
cipitable radioactivity sediments at less than half the rate of the ultraviolet-
absorbing material, which is mostly in 80 S ribosomes. Incubation with
RNAase showed the usual rate of release of nucleotides.

■o

c
o

<u
a.

o
O

1 1 1

• P* c/s

•/

/•

•

•
/o

•

N\a

o Optical density X = 260 —

8
6

°/

o\i\« »

\ •

ON.

1 1 1

Fraction no.

Fig. 7. Growing cells were exposed to P32 for 3 minutes, then broken, and the micro-
some pellet was analyzed in the swinging bucket centrifuge. Note that the maximum
radioactivity does not correspond to the maximum of the ultraviolet absorption. Cf. figure 1.

Those findings, together with its column elution pattern, suggest that the
intermediate is RNA of high molecular weight, either free or associated with
less protein than the bulk of the nucleoprotein. It should be emphasized that
neither lipids nor fragments of cell wall or cell membrane are eluted from the
column, and it is observed that a large part of the P32 incorporated in short ex-
posures is irreversibly bound to the column. An important part of the kinetics
may thereby be missed.

DISCUSSION

To interpret the detailed workings of the cell in terms of its structural com-
ponents, fractionation procedures are needed to separate those components. The

ROBERTS, BRITTEN, AND BOLTON 93

procedures outlined above are only a step toward the needed resolution, but they
have already given indications that:

1. The 80 S ribosomes are composed of nucleoprotein of approximately two
amino acids per nucleotide.

2. This composition is unaltered in the smaller disintegration products re-
sulting from magnesium deficiency.

3. Adsorption followed by elution from the DEAE column causes disintegra-
tion into smaller particles of different composition containing approximately
one amino acid per nucleotide.

4. The protein of the ribosomes is a special protein or at least a special class
of proteins lacking cystine and cysteine. It is therefore doubtful that any of the
enzymes that have been reported in the microsome pellet are actually in the
ribosome fraction.

5. The protein of the ribosomes is most certainly not precursor to the non-
particulate proteins. Such a relationship is ruled out by the data on the com-
position and on the kinetics of formation.

6. Incorporation of amino acids, sulfur, and phosphorus into nucleoprotein of
the ribosomes shows a kinetic delay which indicates that the ribosomes have a
macromolecular precursor.

7. This precursor has properties suggestive of nucleic acid or nucleoprotein
of a low protein content.

These findings when checked and verified will be useful in providing further
conditions that must be met by any theory of protein synthesis. The low initial
specific radioactivity found in the ribosome fraction differs markedly from the
high initial specific radioactivity found in the deoxycholate-insoluble part of the
microsome fraction of rat liver [15]. A partial explanation for the difference
may be that the nucleoprotein has been stripped clean of adhering newly formed
protein by the column; it is not a complete explanation, however, because the
nonparticulate protein of the microsome fraction did not have a high initial
specific radioactivity. More likely, the difference arises from the difference in
the growth rates. If the ribosomes furnish the templates for protein synthesis,
and if chains of 150 amino acid residues are produced by the ribosomes, then
each of the 10,000 ribosomes of a coli cell must produce one polypeptide chain
per 10 seconds to give the observed rate of protein synthesis. If one polypeptide
chain adheres to each ribosome, after 4 minutes' exposure to the tracer only 1/24
of the newly formed polypeptide chains would be found still adhering to the
particles. To show kinetic effects in protein synthesis with these rapidly grow-
ing organisms it will be necessary to use much shorter exposures to the tracer.

The synthesis of the particles themselves appears to be a distinctly different
process, as it proceeds at a more leisurely rate. Even after 24 minutes there are
still marked departures from the steady-state distribution. These findings are
compatible with, but certainly do not prove, the idea that the smaller particles
observed in the cell juice are not simply bits broken off during disruption of

MICROSOMAL PARTICLES

the cell, but that they have biological significance and that they may represent
stages in the growth cycle of the particles.

REFERENCES

1. S. E. Luria, M. Delbruck, and T.
Anderson, /. BacterioL, 46, 57 (1943).

2. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochcm. Biophys., 38,
245 (1952).

3. A. Siegel, S. J. Singer, and S. G.
Wildman, Arch. Biochcm. Biophys., 41,
278 (1952).

4. D. Billen and E. Volkin, /. BacterioL,
67, 191 (1954).

5. S. Dagley and J. Sykes, Arch. Bio-
chcm. Biophys., 63, 338 (1956).

6. H. E. Wade and D. M. Morgan, Bio-
chcm. I, 65, 321 (1957).

7. A. B. Pardee, K. Paigen, and L. S.
Prestidge, Biochim. et Biophys. Acta, 23,
162 (1957).

8. E. T. Bolton, B. H. Hoyer, and D. B.
Ritter, paper 3 of this volume.

9. E. A. Peterson and H. A. Sober,
/. Am. Chem. Soc, 78, 751 (1956).

10. Report of the Biophysics Section, De-
partment of Terrestrial Magnetism, Car-
negie Inst. Wash. Year Boo\ 56, p. 118,
Washington, D. C, 1957.

11. S. Dagley, paper 7 of this volume.

12. O. H. Lowry, N. J. Rosebrough, A.
Furr, A. Lewis, and R. }. Randall, /. Biol.
Chem., 193, 265 (1951).

13. R. B. Roberts, P. H. Abelson, D. B.
Cowie, E. T. Bolton, and R. J. Britten,
Studies of Biosynthesis in Escherichia coli,
Carnegie Inst. Wash. Publ. 607, Washing-
ton, D. C, 1955.

14. Report of the Biophysics Section,
Department of Terrestrial Magnetism,
Carnegie Inst. Wash. Year Boo\ 55, p. 110,
Washington, D. C, 1956.

15. W. Littlefield, E. B. Keller, J. Gross,
and P. C. Zamecnik, /. Biol. Chem., 217 ,
111 (1955). See also J. Brachet, Biochemi-
cal Cytology, pp. 4M7, 240-286, 296-355,
Academic Press, New York, 1957.

Microsomal Structure and Hemoglobin
Synthesis in the Rabbit Reticulocyte

HOWARD M. DINTZIS HENRY BORSOOK JEROME VINOGRAD

Gates and Crellin Laboratories of Chemistry

and Kercl^hoff Laboratories of Biology

California Institute of Technology

A great deal of evidence has now accumulated which suggests very strongly
that microsomal particles are somehow connected with the process of protein
synthesis [1, 2, 3]. Because of their high content of ribonucleic acid which
might act as a coding template, it has become fashionable to postulate that these
particles are the actual sites of protein assembly from activated single amino
acids. To date, however, no evidence has been put forth which could be called
direct proof that such is indeed the case.

Nevertheless, the hypothesis is so attractive that it seems worth while to pro-
ceed on the assumption that it is true and to investigate the detailed structure
of microsomal particles and the relation of this structure to the protein which
the particles are supposedly synthesizing. Such a study would have as its object
an understanding of the molecular nature of the microsomal particle, the molec-
ular structure of the growing peptide chains of the protein being synthesized,
and, if possible, the interrelation between the two.

To best carry out such a study, one would like to find a system consisting of
free floating cells of a single type, actively engaged in the synthesis of predomi-
nantly a single type of protein molecule. Fortunately these desirable properties
are exhibited by mammalian reticulocytes. Such cells can be made in quantity
if rabbits are made anemic by daily injections of phenylhydrazine. After a
week of such injections, reticulocytes (immature red cells) account for 80 to 90
per cent of the red cells present in the blood. These cells are actively producing
hemoglobin and will continue to do so for many hours if suspended in the

96 MICROSOMAL PARTICLES

proper incubation medium [4, 5]. During such incubation, hemoglobin ac-
counts for over 90 per cent of the protein produced.

The microsomal fraction of rabbit reticulocytes has been shown to be very
active in incorporating radioactive amino acids [6]. The following work repre-
sents a beginning step toward an understanding of the relationship between
microsomal structure and hemoglobin synthesis in the sense described above.

SOME PROPERTIES OF MICROSOMAL PARTICLES

Preliminary experiments on isolation of the microsomal fraction showed that
the predominant component had a sedimentation coefficient of about 80 S. Vari-
ations in the method of breaking the cells and in the buffer used for isolating
the microsomal particles were explored before the following standard procedure
was developed. The reticulocytes were frozen, thawed, and mixed with 3 vol-
umes of cold buffer containing 0.14 M KCl, 0.001 M MgCl2, and 0.01 M tris-
chloride, pH 7.2. Cell walls and debris were spun out at low speed. Micro-
somes were then pelleted at 100,000g for 3 hours. The red pellet thus obtained
was then twice redissolved and respun in 100 volumes of buffer, giving a light
amber-colored pellet after the third centrifugation.

Microsomal particles prepared in this way were found to give three compo-
nents in the ultracentrif uge : 82 per cent of 78 S, 9 per cent of 120 S, and 9 per
cent of 50 to 60 S. On electrophoresis in the same buffer the preparation showed
a negative charge, and migrated with only slight skewing in the descending
limb and a splitting into two components in the ascending limb.

The intrinsic viscosity was found to be 0.08 dl/g in the buffer, and the partial
specific volume was found to be 0.63 ml/g.

From these numbers it may be calculated that, if the particles are spherical,
the hydration is 2.6 g of water per gram of anhydrous particle, a very high
value indeed. Taking as a model a highly hydrated sphere, one calculates a
frictional coefficient of 1.72 and a molecular weight of 4.1 XlO6.

That such a model cannot be very far wrong was indicated by light-scatter-
ing measurements on the same preparations. These showed a molecular weight
of 4 XlO6 and a measured dissymmetry of 1.08 (45°/135°) with light of the
mercury blue line. Since a dissymmetry of 1.06 is to be expected for a hydrated
sphere of this molecular weight (diameter, 340 A), the axial ratio cannot be far
from unity, and shapes such as rods and random coils are definitely excluded.

The protein/RNA ratio for these particles was found to be almost unity on
a weight basis. No lipid could be extracted. If the RNA from these particles
is banded in an equilibrium density gradient [7] in cesium formate, the molec-
ular weight of the RNA is found to be approximately 500,000. This is con-
sistent with a small integral number of RNA molecules per microsomal par-
ticle. If the above molecular weights are accepted, this integral number is 4.

DINTZIS, BORSOOK, AND VINOGRAD

BIOLOGICAL ACTIVITY OF MICROSOMAL PARTICLES

In the following experiments rabbit reticulocytes were centrifuged several
times in buffer, suspended at 37° in a medium containing amino acids, iron,
and other materials necessary to ensure optimal hemoglobin syntheses. A given
C14 labeled amino acid was added, and the living cells were incubated for a
definite time period; then several volumes of ice-cold saline were added and the
cells centrifuged several times from cold saline to remove extracellular label.
The cells were then frozen, and microsomes were prepared as described above.

When the cells were incubated for various time intervals with carboxyl C14
labeled leucine (15,500 cpm/mg) the resultant specific activities of protein in
microsomes and in hemoglobin were as shown in table 1.

It would appear that a steady-state concentration of labeled amino acids in
the microsomes is reached within approximately 10 minutes or less.

Incubation
Time,
min.

240

TABLE 1

Specific Activity,

cpm/mg protein

Microsomes

Hemoglobin

0.02

0.01

0.6

0.11

1.1

1.7

4.3

1.8

12.3

3.1

34.9

Rate of Activity

Increase in
Hemoglobin,
cpm/mg/min

0.11

0.22
0.29
0.21
0.15

The labeled amino acid in the purified microsomes is present in some tightly
bound form, as shown by the facts that (1) it is not removed at all by dissolv-
ing the microsomes in buffer saturated with nonlabeled leucine and (2) little,
if any, count is removed by extraction with trichloroacetic acid.

However, the label is rapidly turned over in the living cell, as shown by the
fact that if cells are labeled for 15 minutes in medium containing radioleucine,
and then placed in medium containing nonlabeled leucine for 15 minutes, 90
per cent of the label is removed from the microsomes. This shows that most
of the label present in the microsomal particle is in a dynamic state, and is not
a permanent part of the microsome structure.

In order to obtain information concerning the amino acid composition of the
transient material in the microsome, experiments were conducted using various
labeled amino acids in the incubation medium. Radioleucine was always run
as a control, and the molar ratios of other amino acids to leucine were deter-
mined in the hemoglobin and microsomes after 15 minutes of incubation. The
results are summarized in table 2, where the labeled amino acid ratios are com-
pared with the total amino acid ratios determined by microbiological assay. It
can be seen that the leucine-histidine ratio of transient material in the micro-
some is compatible with the supposition that this material is hemoglobin pro-
tein and not microsome structural protein. The leucine-phenylalanine ratio is

98 MICROSOMAL PARTICLES

not very informative, and the measured leucine-arginine ratio, which lies almost
exactly half way between microsomal protein and hemoglobin, is very ambigu-
ous. These results are compatible with the assumption that the transient ma-
terial in the microsome is largely pre-hemoglobin. More evidence is needed to
confirm this assumption, however.

TABLE 2. Molar Ratios of

Leucine

to V;

inous Amino Acids

Microsomes

Hemoglobin

Total

Composition

Label

Composition

Label

Histidine

3.7

2.2

1.8

1.9

Phenylalanine 2.5

2.1

2.3

2.0

Arginine

1.0

3.2

5.7

7.5

Boiling microsomes with 66 per cent ethanol extracted about 25 per cent of the
radioactive material, whose free amino nitrogen increased greatly on renuxing
with 6 N HC1, suggesting that this material is peptide in nature. The specific
activity of the material was 5 to 10 times that of the unextracted microsomal
protein. Both the extractable and nonextractable radioactive materials were
transient; i.e., the counts were removed on incubating cells in nonlabeled amino
acid for 15 minutes.

One would like to conclude from the above extraction data that the extracted
material is richer than the whole microsome in growing peptide chains of short
length, whereas the unextractable material represents growing peptide chains
which are too long to dissolve in 66 per cent ethanol. The short chains would
presumably represent the earliest stages of hemoglobin formation in the micro-
some. Further purification and characterization are necessary to prove this
point.

SOME NUMEROLOGY AND CONCLUSIONS

The above data lead to some interesting results if the following assumptions
(or approximations) are made: (1) hemoglobin is the only protein being made
in rabbit reticulocytes; (2) all microsomal particles are equally active in syn-
thesizing hemoglobin; (3) all the transient label in the microsome is pre-hemo-
globin. The steady-state label level of the microsomal particle, the specific ac-
tivity of leucine used, together with the facts that 12 per cent of the protein is
leucine and one-half of the microsome is protein, lead to the conclusion that
0.05 per cent of the mass of the microsomal particle is pre-hemoglobin, i.e., grow-
ing peptide chain. Since the molecular weight of the microsomal particle is
4,000,000 as shown above, this means that the average weight of growing chain
per particle is 2000. In a random population of growing chains the average
weight might be expected to be about one-half of the finished chain weight. If
all the growing chain per particle is in one piece, this leads to a value of 4000
for the finished weight of polypeptide chain made per microsomal particle, a
value reasonably close to the weight of one-fourth of a hemoglobin molecule,

DINTZIS, BORSOOK, AND VINOGRAD 99

i.e., one polypeptide chain. If assumption 2 above is incorrect, and only a frac-
tion of the microsomal particles is functional, the agreement is even better.
From the rate of incorporation of label into finished hemoglobin molecules and
the concentrations in the living cells of hemoglobin (15 per cent) and micro-
somes (0.5 per cent), one may calculate that to account for the production of
new hemoglobin each microsomal particle must make one-quarter of a hemo-
globin in 1.5 minutes.

SUMMARY

The above data lead to the picture of a microsomal particle as an almost spher-
ical sponge-like structure of anhydrous molecular weight 4,000,000 and diame-
ter 340 A. One-half of the mass is represented by ribonucleic acid which ap-
pears to be present as four strands of molecular weight 500,000. The half of
the microsomal particle which is protein appears to be almost entirely (99.9 per
cent) structural in nature; i.e., it is not transient protein precursor.

Woven into this sponge-like structure in some way is a very small amount
(0.05 per cent by weight) of transient protein precursor. Taken together with
the observed rate of hemoglobin production, this amount of precursor is com-
patible with the conclusion that one microsomal particle makes one polypeptide
chain of hemoglobin in approximately 1 minute.

Contribution No. 2338

REFERENCES

1. H. Borsook, C. L. D:asy, A. J. 4. J. Kruh and H. Borsook, /. Biol.

Haagen-Smit, G. Keighley, and P. H. Chem., 220, 905 (1956).

Lowy, /. Biol. Chan., 187, 839 (1950). 5. H. Borsook, E. H. Fischer, and G.

2. J. W. Littlefield, E. B. Keller, J. Gross, Keighley, /. Biol. Chan., 229, 1059 (1957).

and P. C. Zamecnik, /. Biol. Chan., 217, 6. M. Rabinovitz and M. E. Olson,

111 (1955). Exptl. Cell Research. 10, 747 (1956).

3. J. W. Littlefield and E. B. Keller, 7. M. Meselson, F. W. Stahl, and J.

/. Biol. Chan., 224, 13 (1957). Vinograd, Proc. Natl. Acad. Sci. U. S., 43,

581 (1957).

Effects of ^-Fluorophenylalanine on the
Growth and Physiology of Yeast1

G. N. COHEN H. O. HALVORSON

Department of Bacteriology, University of Wisconsin

S. SPIEGELMAN
Department of Bacteriology, University of Illinois

Halvorson and Spiegelman [1] examined a series of amino acid analogs for
their ability to inhibit growth of yeast, to deplete the "free amino acid pool,"
and to synthesize protein. When exponentially growing yeast is washed and
resuspended in a nitrogen-free buffer in the presence of glucose, the free amino
acids are rapidly incorporated into proteins, thus depleting the amino acid pool.
In the presence of high concentrations of ^-fluorophenylalanine (10-2 M to
2X10"2 M), this depletion is severely inhibited, as shown by chromatographic
study of the pool components or by analysis of the glutamic acid content of the
pool. This inhibition was interpreted as follows: The presence of an amino
acid analog prevents the incorporation not only of its natural homolog but of all
the other amino acids as well.

On the other hand, Munier and Cohen [2] found that /7-fluorophenylalanine,
when added to an exponentially growing culture of Escherichia coli, caused a
linear growth. During that period the differential rate of incorporation of
valine or S35

(Avaline or AS35) /Amass

was the same as in a control culture, although incorporation was somewhat
slower than during exponential growth. Radioactive amino acids were formed

1 This investigation was aided by a grant (G-4258) from the Division of Biological and
Medical Sciences of the National Science Foundation.

100

COHEN, HALVORSON, AND SPIEGELMAN 101

from radioactive glucose and incorporated in the presence of the analog during
the linear growth. In addition, /7-fluorophenylalanine was incorporated (Munier
and Cohen [2], [3]) to a great extent (up to 200 m|jmoles/g dry weight). In-
duced (3-galactosidase was synthesized at the same differential rate as in a con-
trol culture without analog. Because of the contradictory results of these two
studies, it was decided to re-examine the effects of /7-fluorophenylalanine on
yeast.

MATERIALS AND METHODS

Saccharomyces italicus Y1225 was used in these experiments. The cultures
were grown in synthetic medium (Halvorson and Spiegelman [1]) in Erlen-
meyer flasks which were shaken at 30° C. During the exponential phase of
growth, the protein content per cell was found to be proportional to the opti-
cal density. Therefore, for reasons of convenience, growth was followed by
measurements of the optical density in a Beckman DU Spectrophotometer at
600 mu. Under these conditions 1.00 O D = 772 ug dry wt./ml.

Viable counts were determined by plating appropriate dilutions on dextrose
broth agar medium.

The cells were centrifuged, washed, and fractionated for isotope distribution
as previously described (Halvorson [4]). The components of the protein hy-
drolysates were identified by radioautography. For radioactivity measurements,
aliquots were evaporated to dryness on stainless-steel planchets and counted in
a gas flow counter, and the radioactivities were corrected to infinite thinness.

The photomicrographs were taken on a 35-mm microfile film with a 100
apochromatic objective, N. A. = 1.30. Magnification on the film was 1250 X.
3-C14-DL-phenylalanine (Phe) (2.1 mc/mmole), 4-4'-C14-DL-valine (1.33 mc/
mmole), and 3-C14-DL-p-fluorophenylalanine (/>-FPhe) (2.35mc/mmole) were
obtained from the Commissariat a l'Energie Atomique, France. Uniformly
labeled glucose (2.4 mc/mmole) was obtained from the Fisher Scientific Com-
pany, and carrier-free S35 sulfate from the Oak Ridge National Laboratory.

RESULTS

Effects of p-Fluorophenylalanine and $-2 Thienylalanine on the Growth of
Yeast. Figure 1 shows that, upon addition of 10 2 M /7-FPhe or $-2 thienyl-
alanine (Thiala) to an exponentially growing culture of yeast, the mass in-
creases at a rate which is linear with time, as previously observed with E. coli.
In this particular experiment, from the time of addition to the termination of
growth, the mass increased 4 times in the presence of /7-FPhe and 2.7 times in
the presence of Thiala. Under these conditions, although no component of the
medium was limiting for growth, there was no increase in viable count in
the presence of the antagonists. The increase in optical density in the presence
of /7-FPhe (4 times) can be attributed to an increase in cell size (fig. 2). As-
suming that yeast cells are ellipsoids of revolution, the average cell volume in-
creased approximately 3.6 times in the presence of p-FPhe.

102

MICROSOMAL PARTICLES

H-q Dry Weight /ml
200

150

100

p-FPhe

Thiala

120 240

Time (min)

360

Fig. 1. Effects of p-fluorophenylalanine and (3-2-thienylalanine on the growth of yeast.

Synthesis of Cell Material during Linear Growth. Washed cells from an
exponentially growing culture were placed in two flasks, one containing C14
glucose, and the other containing C14 glucose and /?-FPhe (10~2 M final
concentration).

Optical density was followed throughout the experiment, and 10-ml samples
taken at intervals were placed in precooled centrifuge tubes (0° C). The
cells in the control culture were allowed to increase 2.6 times, and those in
the linear culture 1.9 times. The samples were fractionated (Halvorson [4]),
and the radioactivity of the hot-TCA-soluble fraction (containing the nucleic
acids) and of the protein fraction (hot-TCA-insoluble) was determined. The
differential rates of synthesis of total hot-TCA-soluble material and of total pro-
tein were then plotted. The differential rate of synthesis of a component X is
expressed by AX/AM, M being the increase in mass during the time necessary

Fig. 2. Effect of /7-fluorophenylalanine on the size of yeast cells. Pictures taken initially
(.i), after an optical density increase of 2.7 times (B), and 5.4 times (C).

COHEN, HALVORSON, AND SPIEGELMAN

103

to obtain the increase AX; this expression is independent of the rate of growth.
Figures 3 and 4 show that the differential rates of synthesis of hot-TCA-soluble
material and of protein are the same in the presence and absence of p-FPhe.

/ig Hot TCA Soluble C/m

H-t Protein C/m

o control
• p-FPhe

100 200

eg Dry Weight/ml

100 200

CO Dry Weight /ml

Fig. 3. Differential rate of synthesis of
total hot-TCA-soluble material in the pres-

Fig. 4. Differential rate of synthesis of
proteins in the presence or absence of

ence or absence of p-fluorophenylalanine. See p-fluorophenylalanine. See text for details.
text for details.

Incorporation of Cli-p-Eluorophenylalanine in Exponentially Growing Yeast.
Radioactive /7-FPhe was added to two exponentially growing cultures at final
concentrations of 3.87 X 10 4 M and 1.04 X 10"3 M, and the cultures were allowed
to grow linearly (300 minutes) until their mass increased by 3.6 times. Samples
were taken at given intervals, and the radioactivity of the protein fraction was
determined. The differential rate of incorporation of /7-FPhe was calculated,
and from the slopes of the straight lines obtained (fig. 5) the content of /7-FPhe
in the proteins was found. As in E. coli, increasing the external concentrations
of p-FPhe increases the analog content of the proteins. The incorporation is
far from negligible, reaching 12 per cent of the normal phenylalanine content
for a concentration of the analog of 1.03 XlO"3 M (31.2 umoles /7-FPhe/g dry
wt.). Munier (personal communication) and Kerridge (quoted in Gale and
McQuillen [5]) have also shown that /7-FPhe is incorporated into yeast proteins.
The relative amount of phenylalanine in yeast was determined from its differ-
ential rate of incorporation. In two experiments, where the C14 phenylalanine
concentration was 3.12 X 10"5 M and 6.25 X 10~5 M respectively, identical differen-
tial rates of incorporation were observed.

In all experiments, radioautograms of the acid hydrolysates of the protein
fractions were made. Both for cells grown on C14-Phe and for those grown on
C14-/7-FPhe, the radioactivity of the protein hydrolysates was identified exclu-
sively with the added isotope.

104

MICROSOMAL PARTICLES

m/i moles Protein p-FPhe /ml
1

3 87-10" M

100 150

eg Dry Weight/ ml

0 4

0. 3

0.2

O.I

Ht) Protein S/ml

o control
• p-FPhe

20 30

Time ( mln)

Fig. 5. Differential rate of incorporation Fig. 6. Effect of p-fluorophenylalanine

of p-fluorophenylalanine. See text for details. on S35 sulfate incorporation into proteins of

resting yeast cells. See text for details.

Incorporation of p-Fluorophenylalanine under "Resting" Conditions. Since
the experiments of Halvorson and Spiegelman [1] were conducted with "rest-
ing" cells, it was interesting to know whether incorporation of p-FPhe occurred
also under these conditions. Exponentially growing yeast was washed twice
with buffer and resuspended in phosphate-succinate buffer, pH 4.7, with glu-
cose as energy source, but without an exogenous nitrogen source. The sus-
pension was divided in two flasks to which C14 Phe and C14 p-FPhe were
respectively added (final concentration: 6.25x10 3 M). In both flasks the in-
corporation of radioisotope was linear with time and ceased after 50 minutes'
incubation. At this time, 31.8 mumoles of Phe had been incorporated, whereas
19.4 mumoles of p-FPhe had been incorporated in the proteins per milliliter
of culture. Thus, the analog was incorporated at 61 per cent the extent of Phe
incorporation.

Incorporation of Sulfur from Radiosulfate into Yeast Proteins under "Rest-
ing" Conditions. An exponential culture from synthetic medium was cen-
trifuged, washed, resuspended in nitrogen-free medium, and divided in two
flasks containing S35 with and without 0.01 M p-FPhe. The incorporation of
S35 into proteins was linear with time over 40 minutes with identical slopes of
incorporation in the two flasks (fig. 6).

Incorporation of Radiovaline under "Resting" Conditions in Presence or
Absence of p-FPhe. Yeast was grown in broth, centrifuged while in the ex-

COHEN, HALVORSON, AND SPIEGELMAN

105

ponential phase, washed twice, and resuspended in phosphate-succinate buffer
with glucose but without an exogenous source of nitrogen. Radioactive valine
was added at 0 time, and the suspension was shaken for 8 minutes at 30° C.
The suspension was then centrifuged, washed twice in the cold, and divided
into two flasks in the same medium without valine, and with and without
10"2 M p-FPhe. Valine incorporation proceeded at the same rate in the two
flasks (fig. 7) ; as shown previously (Halvorson and Cohen [6]), the valine pool
size is sufficient under these conditions for an unchanged rate of valine incor-
poration. These experiments show that, under growing or "resting" condi-
tions, p-FPhe does not inhibit protein synthesis. It was then interesting to
find out why, under the conditions of Halvorson and Spiegelman [1], the pools
were not depleted in the presence of p-FPhe.

m/imoles Protein Valine/ml
I4r

"iMfl Protein S/ ml
3O0OT

200C-

» NH,

1000

10 12

Time (mln)

Fig. 7. Valine incorporation into the pro-
teins of resting yeast cells. See text for
details.

a p-FPhe

100 200

Time(min)

Fig. 8. Effect of exogenous nitrogen on
the incorporation of S35 into proteins in ni-
trogen-starved cells. See text for details.

The Effect of Nitrogenous Compounds on the Incorporation of Sulfur into
Proteins in Nitrogen-Starved Cells. A culture growing exponentially in broth
was centrifuged, resuspended with glucose in a phosphate-succinate buffer, and
then shaken for 18 hours (original dry weight: 1.55 mg/ml). These starved
cells were then washed and resuspended in nitrogen-free, phosphate-succinate
buffer with glucose and S35 sulfate with or without 10~2 M NH4C1, 10-2 M Phe,
or 10"2 M p-FPhe. The curves of figure 8 show that /7-FPhe and Phe can act as
nitrogen sources for S incorporation, probably providing — NH2 groups through
transamination to carbon acceptors.

Effect of p-Fluorophenylalanine on Protein Degradation. In the absence of
an exogenous source of nitrogen and energy, the degradation of cellular proteins
leads to elevated pool levels (Halvorson [7]). Since high amino acid pool levels

106

MICROSOMAL PARTICLES

are observed in the presence of p-FPhe (Halvorson and Spiegelman [1]), a
direct test of its effect on the rate of protein degradation was undertaken.

Cells were grown overnight in synthetic medium in the presence of radio-
active valine or Phe. They were then centrifuged, washed, resuspended in fresh
synthetic medium containing glucose, and shaken for 210 minutes in order to
diminish any pool radioactivity that might have been present. The cells were
again centrifuged, washed twice, and resuspended to a density of 3 mg dry
wt./ml in two flasks containing buffer with or without p-FPhe 10~2 M. Protein
degradation was followed by the appearance of radioactivity in the soluble pool
(cold-TCA-soluble radioactivity). The results in table 1 show elevated pool
levels in the presence of p-FPhe. Since the previous experiments show that
p-FPhe does not inhibit the amino acid incorporation observed in the presence
of an exogenous source of energy, these results indicate that the antagonist ac-
celerates the rate of protein degradation.

TABLE 1. Effect of p-Fluorophenylalanine on Protein Breakdown

Radioactivity Released f

Incubation time,*

Growth Supplement

min

C14 valine X

120

180

240

C11 phenylalanine §

120

180

240

Control

40
145
270

54
171
378
613

p-FPhe

90
210
380
870
463
870
1460
2310

* Incubated aerobically in phosphate buffer, pH 4.5, at 30° C with or without 0.01 M
p-FPhe.
t Increase in cpm of cold-TCA-soluble fraction/ml incubation mixture.
t 19,860 cpm protein/ml incubation mixture.
§ 81,320 cpm protein/ml incubation mixture.

DISCUSSION

A reanalysis of the effects of /7-FPhe on the growth of yeast shows a strong
parallelism with its effects on E. coli. A linear rather than exponential rate
of growth is seen in the absence of cell division and without decreasing the dif-
ferential rates of synthesis of protein and hot-TCA-soluble material or total
carbon incorporation. Furthermore, in resting yeast cells, the incorporation of
endogenous amino acids is not influenced by the presence of p-FPhe. The previ-
ously observed inhibition of a-glucosidase synthesis by p-FPhe (Halvorson and
Spiegelman [1]) may therefore represent another example of inactive enzyme
synthesis (Cohen and Munier [8]). In contrast to the results found with E. coli,
however, p-FPhe is capable of completely suppressing a-glucosidase induction
in resting yeast cells only when added simultaneously with the inducer (Hal-
vorson and Jackson [9]). When p-FPhe was added at various times after the

COHEN, HALVORSON, AND SPIEGELMAN 107

inducer, induced synthesis became more and more refractory to the inhibitor.

One feature of the effect of /7-FPhe on resting yeast cells requires special
attention. Previous studies showed that, in the presence of /7-FPhe, the contents
of the free amino acid pool remained essentially the same as in unstarved cells
(Halvorson and Spiegelman [1]). On the basis of the present experiments, the
nondisappearance of the pool in the presence of p-FPhe can be attributed to:
(1) partial replenishment of the free amino acid pool from the nitrogen of
/7-FPhe, and (2) an increased rate of protein degradation in the presence of
/7-FPhe.

The reversal of /7-FPhe inhibition by Phe can be related not only to a com-
petition between these two amino acids for an accumulating system (Halvor-
son and Cohen [6]) but also to its incorporation into proteins. Halvorson and
Spiegelman [1] had derived from their experiments with /7-FPhe the conclu-
sion that there were no intermediate precursors of induced maltozymase in
yeast. Although amino acid antagonists may prove valuable for studies on
protein synthesis, it would seem on the basis of these and other experiments
(Cohen and Munier [8]) that conclusions derived from their use as a tool in
the study of intermediates in protein synthesis are unwarranted. The de novo
nature of induced enzyme synthesis has been established on other grounds,
however (Hogness, Cohn, and Monod [10]; Rotman and Spiegelman [11]).

ACKNOWLEDGMENTS

We wish to express our appreciation to Dr. R. Munier for advice and assist-
ance in the early phases of these experiments and to Dr. Pichat of the Com-
missariat a l'Energie Atomique, France, for the synthesis of 3-C14-DL-/7-fluoro-
phenylalanine.

SUMMARY

The addition of 0.01 M /7-fluorophenylalanine to a growing culture of Sac-
charomyces italicus Y1225 results in (1) an incorporation of the analog into
cellular proteins, (2) a linear rather than an exponential rate of growth as a
function of time, and (3) an inhibition of cell division. The antagonist does
not influence the differential rates of synthesis of protein and hot-TCA-soluble
material.

Under "resting conditions," p-fluorophenylalanine does not inhibit either pro-
tein synthesis or the utilization of the free amino acid pool. Elevated pools in
the presence of the antagonist were attributed to pool replenishment from the
nitrogen of antagonist and to an increased rate of protein degradation.

REFERENCES

1. H. O. Halvorson and S. Spiegelman, Incorporation d'analogues structuraux
The inhibition of enzyme formation by d'amino acides dans les proteines bacterien-
amino acid analogues, /. BacterioL, 64, 207 nes, Biochim. et Biophys. Acta, 21, 592-
(1952). 593 (1956).

2. R. L. Munier and G. N. Cohen, 3. R. L. Munier and G. N. Cohen,

108

MICROSOMAL PARTICLES

Incorporation d'analogues structuraux
d'amino acides dans les proteines d'Esch-
erichia coli, Ann. inst. Pasteur, in press.

4. H. O. Halvorson, Studies on protein
and nucleic acid turnover in growing cul-
tures of yeast, Biochim. et Biophys. Acta,
27, 267 (1958).

5. E. F. Gale and K. McQuillen, Nitro-
gen metabolism, Ann. Rev. Microbiol., 11,
283 (1957).

6. H. O. Halvorson and G. N. Cohen,
Incorporation comparee des amino acides
endogenes et exogenes dans les proteines
de la levure, Ann. inst. Pasteur, in press.

7. H. O. Halvorson, Intracellular protein
and nucleic acid turnover in resting yeast
cells, Biochim. et Biophys. Acta, 27 , 255
(1958).

8. G. N. Cohen and R. L. Munier, Effect
des analogues structuraux d'amino acides
sur la croissance, la synthase de proteines
et la synthese d'enzymes, chez Escherichia
coli, Ann. inst. Pasteur, in press.

9. H. O. Halvorson and L. Jackson, The
relation of ribose nucleic acid to the early
stages of induced enzyme synthesis in
yeast, /. Gen. Microbiol., 14, 26 (1956).

10. D. Hogness, M. Colin, and J. Monod,
Induced synthesis of beta-galactosidase in
E. coli, Biochim. et Biophys. Acta, 16, 99
(1955).

11. B. Rotman and S. Spiegelman, On
the origin of the carbon in the induced
synthesis of beta-galactosidase in Escher-
ichia coli, ]. Bacterial., 68, 419 (1954).

Enzymatic and Nonenzymatic Synthesis in

Adenyl Tryptophan1

MARVIN KARASEK PAUL CASTELFRANCO 2

P. R. KRISHNASWAMY ALTON MEISTER

Department of Biochemistry, Tufts University School of Medicine

Acyl adenylates have been postulated as intermediates in the activation of
acetate [1] and of fatty acids [2, 3], as well as in the synthesis of phenylacetyl-
glutamine and hippurate [4]. Thus, the activation of acetate and of phenylace-
tate may be represented as follows: 3

Acetate + ATP ±± Acetyl-AMP + PP
Phenylacetate + ATP ^± Phenylacetyl-AMP + PP

The activation of amino acids, which has been observed with several enzyme
preparations, appears to involve an analogous reaction [5, 6, 7, 8] :

Amino acid + ATP *± Aminoacyl-AMP + PP

This reaction has been followed by observing the formation of amino acid hy-
droxamate when enzyme preparations are incubated with amino acid, mag-
nesium ions, adenosine triphosphate, and high concentrations of hydroxylamine.
The reaction has also been observed by determining the rate of exchange of
radioactive inorganic pyrophosphate with adenosine triphosphate in the pres-
ence of magnesium ions, amino acid, and enzyme.

That the intermediate formed in the activation of amino acids is an aminoacyl
adenylate of the type (fig. 1) postulated to occur in other systems [1-4] is sug-
gested by several observations. For example, synthetic acyl adenylates are

1 Supported in part by research grants from the National Science Foundation and the
National Institutes of Health, Public Health Service.

2 Postdoctorate fellow of the National Heart Institute, Public Health Service.

3 Abbreviations: adenosine triphosphate, ATP; adenylic acid, AMP; pyrophosphate, PP.

109

110 MICROSOMAL PARTICLES

HO 0

I || II

- CH2 -C-C - 0- P-O- Ribose -Adenine"1"
I l

NH3+ 0~

Fig. 1. Tryptophanyl adenylate.

known to react promptly with hydroxylamine to yield the corresponding hy-
droxamates [1-4]. Furthermore, it has been reported that synthetic leucyl
adenylate formed adenosine triphosphate when incubated with inorganic pyro-
phosphate and an activating enzyme purified from Escherichia coli [8]; a simi-
lar experiment has been carried out with methionyl adenylate and an activat-
ing enzyme isolated from yeast [6]. An additional piece of evidence consistent
with the formation of an anhydride linkage between the phosphoric acid
group of adenylic acid and the carboxyl group of amino acids has been ob-
tained in experiments with amino acids labeled with O18; transfer of O18 from
the carboxyl group of the amino acid to adenylic acid was associated with
enzymatic activation. The pyrophosphate formed did not contain appreciable
quantities of O18 [9].

The available data are consistent with the hypothesis that aminoacyl adenyl-
ates are intermediates in the amino acid activation reaction, but the formation
of such anhydrides has not yet been shown. Although there is as yet no experi-
mental demonstration of the net synthesis of acyl adenylates in acetate or fatty
acid oxidation, the respective enzyme systems are apparently able to utilize
added synthetic acyl adenylate derivatives [1-3]. Previous inability to detect
the formation of such intermediate anhydrides may be related to the instability
of the anhydrides and perhaps also to the high affinity of the enzyme for the
anhydride; accordingly the actual intermediate in these activation reactions
may be enzyme-bound acyl adenylate.

We have attempted to obtain evidence for the net synthesis of tryptophanyl
adenylate by the tryptophan-activating enzyme of beef pancreas [7]. In these
studies, we have used aminoacyl adenylates prepared as described in the fol-
lowing paper by Castelfranco et al. [10]. Although tryptophanyl adenylate is
hydrolyzed rapidly at pH 7.2 and 37° C, we have found that only about 10 per
cent of the anhydride is hydrolyzed in 2 hours at pH 4.5 and 0°. Paper
ionophoresis at pH 4.5 in 0.05 M ammonium formate buffer at 0° indicated
that tryptophanyl adenylate was positively charged and moved with a greater
mobility than tryptophan itself (fig. 2) . The positively charged band quenched
the fluorescence of the paper under ultraviolet light and gave the ninhydrin
color reaction. Elution of this material from the paper strip yielded an alkali-
labile compound which formed a hydroxamic acid promptly on treatment with
hydroxylamine. The hydroxamate was identified as tryptophan hydroxamate
by paper chromatography in several solvent systems. Incubation of the eluted
compound with the tryptophan-activating enzyme, magnesium chloride, inor-

KARASEK ET AL.

Ill

TRY

TRY- AMP

-6

6 cm

Fig. 2. Paper ionophoresis of tryptophan (TRY) and tryptophanyl adenylate (TRY-
AMP) in 0.05 M ammonium formate buffer (pH 4.5) ; apparatus of Markham and
Smith [11].

ganic pyrophosphate, and tris(hydroxymethyl) aminomethane buffer led to
synthesis of adenosine triphosphate. Adenosine triphosphate was identified by
coupling the reaction between radioactive inorganic pyrophosphate and trypto-
phanyl adenylate with the phosphorylation of glucose by hexokinase. The phos-
phate esters were separated by ethanol-barium salt fractionation, and radioac-
tive glucose-6-phosphate was identified by paper chromatography in two solvent
systems.

An experiment designed to demonstrate the net synthesis of tryptophanyl
adenylate was carried out as follows. The total yield of tryptophan-activating
enzyme obtained from 10 lb of beef pancreas (15 mg) was incubated with
DL-tryptophan-3-C14, magnesium chloride, crystalline pyrophosphatase, adenosine
triphosphate, and tris (hydroxy methyl) aminomethane buffer for 30 minutes
at 37° C. At the end of the incubation period, 2 mg of synthetic tryptophanyl
adenylate was added as carrier and the reaction mixture was lyophilized. The
lyophilized reaction mixture was fractionated according to the scheme shown
in figure 3.

In the first step of this procedure, treatment with glacial acetic acid in the

TRY-C14

ATP +

Mg++

+ Enzyme

Glacial
acetic
acid

J — "

Enzyme ]

ATP + TRY-C14 +TRY-AMP

v Ether

ATP + TRY-AMP

TRY-C

Paper electrophoresis
(/>H 4.5 and 0°)

Fig. 3. Scheme for the isolation of tryptophanyl adenylate (TRY-AMP) from enzymatic
reaction mixtures.

112 MICROSOMAL PARTICLES

cold separated tryptophanyl adenylate from the enzyme which remained in
the insoluble residue along with some magnesium chloride and tris(hydroxy-
methyl) aminomethane buffer. Subsequent addition of ether to the acetic acid
extract resulted in the precipitation of adenosine triphosphate and tryptophanyl
adenylate, leaving radioactive tryptophan in the supernatant solution. The pre-
cipitate, which contained both adenosine triphosphate and tryptophanyl adenyl-
ate, was washed several times with cold ether-glacial acetic acid followed by
several ether extractions in order to remove residual acetic acid. The precipi-
tate was dissolved in a small amount of ammonium formate buffer (pW 4.5),
and an aliquot of this material was analyzed ionophoretically. The paper
ionophoretic separation yielded a major radioactive band which corresponded in
mobility to authentic tryptophanyl adenylate. A smaller band, negatively
charged, was detected but has not yet been identified. The positively charged
band was eluted, treated with hydroxylamine, and chromatographed on paper
in several solvent systems. Radioactive tryptophan hydroxamic acid was identi-
fied in each system.

A similar experiment carried out without the addition of carrier trypto-
phanyl adenylate was also performed. Ionophoretic analysis again revealed evi-
dence for the formation of tryptophanyl adenylate; however, somewhat less
radioactivity was found in the tryptophanyl adenylate area. These findings sug-
gested that there was incorporation of radioactive tryptophan into tryptophanyl
adenylate in the experiment with carrier. In order to investigate this possi-
bility directly, radioactive tryptophan was incubated with tryptophanyl adenyl-
ate, enzyme, and magnesium ions. The tryptophan hydroxamate isolated
from this reaction mixture (after the addition of hydroxylamine) contained
appreciable radioactivity; the findings therefore suggest that an exchange be-
tween tryptophan and tryptophanyl adenylate occurred :

TRY-C1 4 + TRY-AMP -» TRY-C14-AMP 4- TRY

We have also found that the tryptophan-activating enzyme catalyzes the
formation of adenosine triphosphate from inorganic pyrophosphate and a va-
riety of a-aminoacyl adenylates. Thus, aminoacyl adenylates of l- and D-trypto-
phan, l- and D-phenylalanine, L-isoleucine, L-glutamine, L-alanine, glycine, L-pro-
line, L-valine, L-leucine, and L-tyrosine gave adenosine triphosphate in this
system. It is of some interest that d- and L-tryptophanyl adenylate and d- and
L-phenylalanyl adenylate were about equally active. Examination of the D-anhy-
drides (after hydrolysis) by optically specific enzymatic methods [12] revealed
that the optical purity of the amino acid moieties was greater than 99.5 per cent;
it is therefore unlikely that the activity of the D-aminoacyl adenylate is due to
the presence of adenylate derivatives of the corresponding enantiomorphs. Of
the aminoacyl adenylates examined, only those of a-amino acids were active.4

4 We have recently found that L-tryptophanyl inosinate [10] is inactive, and that inosine
triphosphate is not active in place of adenosine triphosphate in the forward reaction (reac-
tion 1, table 2).

KARASEK ET Ah. 113

Thus, carbobenzoxytryptophanyl adenylate, 3_alanyl adenylate, and benzoyl
adenylate were inactive (table 1). Table 2 summarizes some of the reactions
catalyzed by the tryptophan-activating enzyme preparation. Under the condi-
tions employed, the enzyme catalyzed the formation of L-tryptophan hydroxa-
mate, but not that of the d isomer. This is in striking contrast to the results ob-
tained on the synthesis of adenosine triphosphate (table 1). Further studies of
the specificity of the enzyme system are in progress.

TABLE 1. Specificity of the Tryptophan-Activating Enzyme with Respect to Acyl
Adenylate in Synthesis of Adenosine Triphosphate

Acyl Adenylates

A .

I \ \

Active Inactive

L-Tryptophan-AMP 3-Alanine-AMP

D-Tryptophan-AMP Acetyl-AMP

L-Phenylalanine-AMP Benzoyl-AMP

D-Phenylalanine-AMP Carbobenzoxytryptophan-AMP

L-Glutamine-AMP Phenylacetyl-AMP

L-Isoleucine-AMP
L-Leucine-AMP
L-Valine-AMP
L-Alanine-AMP
L-Tyrosine-AMP
Glycine-AMP
L-Proline-AMP
L-Threonine-AMP
L-Serine-AMP

TABLE 2. Types of Reactions Catalyzed by Tryptophan-Activating Enzyme

Specificity

Reaction Type l

(1)

TRY + ATP + NH2OH +

(2)

TRY-AMP + PP +

(3)

l-TRY-AMP + dl-TRY-C14

(4)

Amino acid + ATP + NH2OH 0

(5)

Amino acid-AMP + PP +

Studies

with d- and l-TRY-C14 are not yet complete.

D
0

Note Added in Proof

Novelli [Proc. Natl. Acad. Set. U. S.t 44, 86 (1958)] has very recently reported
synthesis of adenosine triphosphate from pyrophosphate and several aminoacyl
adenylates with this enzyme, and Berg {Federation Proc., 16, 152 (1957) ; per-
sonal communication] has made similar observations with a methionine-acti-
vating enzyme obtained from yeast. Rhodes and McElroy (personal communi-
cation) have recently observed enzymatic synthesis of adenyl oxyluciferin by

114

MICROSOMAL PARTICLES

firefly luciferase; they have also obtained evidence for tight binding of this
intermediate to the enzyme.

Recent studies in our laboratory indicate that the affinity of the tryptophan-
activating enzyme is greater for L-tryptophanyl adenylate than for D-trypto-
phanyl adenylate and a number of the other ot-aminoacyl adenylates listed in
table 1.

REFERENCES

1. P. Berg, /. Biol. Chen?., 222, 1015
(1956).

2. W. P. Jencks and F. Lipmann, /. Biol.
Chem., 225, 207 (1957).

3. H. S. Moyed and F. Lipmann, /.
Bacterial., 73, 117 (1957).

4. K. Moldave and A. Meister, /. Biol.
Chem., 229,463 (1957).

5. M. B. Hoagland, E. B. Keller, and
P. C. Zamecnik, /. Biol. Chem., 218, 345
(1956).

6. P. Berg, /. Biol. Chem., 222, 1025
(1956).

7. E. W. Davie, V. V. Koningsberger,

and F. Lipmann, Arch. Biochem. Bio-
phys., 65, 21 (1956).

8. J. A. DeMoss and G. D. Novelli, Bio-
chim. et Biophys. Acta, 22, 49 (1956).

9. M. B. Hoagland, P. C. Zamecnik, N.
Sharon, F. Lipmann, M. P. Stulberg, and
P. D. Boyer, Biochim. et Biophys. Acta, 26,
215 (1957).

10. P. Castelfranco, A. Meister, and
K. Moldave, paper 14 of this volume.

11. R. Markham and J. B. Smith, Bio-
chem. J., 52, 552 (1952).

12. A. Meister, L. Levintow, R. M.
Kingsley, and J. P. Greenstein, /. Biol
Chem., 192, 535 (1951).

Participation of Adenyl Amino Acids in Amino
Acid Incorporation into Proteins1

PAUL CASTELFRANCO - ALTON MEISTER KIVIE MOLD AVE

Department of Biochemistry, Tufts University School of Medicine

It has been postulated that aminoacyl adenylates possessing the general struc-
ture shown in figure 1 are formed in the enzymatic activation of amino acids

HO 0
i ii ii

R-C-C-O-P- 0-Ribose-Adenine +

I i

NH3+ 0"

Fig. 1

by adenosine triphosphate, and that such "activated amino acids" are inter-
mediates in the incorporation of amino acids into microsomal proteins observed
in cell-free systems [1, 2]. Whether such incorporation represents protein syn-
thesis is not yet known, although this hypothesis has indeed been considered.
Recent studies in our laboratory have been directed toward a better under-
standing of the activation of amino acids and the possible role of aminoacyl
adenylates in amino acid incorporation into protein. In the preceding paper
by Karasek et al. [3], evidence for the net synthesis of tryptophanyl adenylate
by a purified tryptophan-activating enzyme is described. These observations
appear to give direct support to the idea that aminoacyl adenylates are the
initial products of the activation reaction. The subsequent reactions of ami-
noacyl adenylates are not yet clearly understood, although it has been sug-
gested that the amino acid moieties of such anhydrides are transferred to pro-
tein via specific acceptors, possibly ribonucleic acid [4].

1 Supported in part by research grants from the National Science Foundation and the
National Institutes of Health, Public Health Service.

2 Postdoctorate fellow of the National Heart Institute, Public Health Service.

115

116 MICROSOMAL PARTICLES

We began this work by attempting to prepare a number of aminoacyl adenyl-
ates in a reasonable state of purity and in good yield in order to make possi-
ble the synthesis of radioactive aminoacyl adenylates. It is not unusual in mod-
ern biochemical research to synthesize and study compounds that are believed
to be intermediates in biochemical reactions. In the present instance this ap-
proach may suffer from a possible difficulty in that the intermediates may be
bound to enzymes and therefore not be in equilibrium with an external source
of intermediate. It has been observed, however, that chemically synthesized acyl
adenylate derivatives are enzymatically active in systems that catalyze activa-
tion of acetate [5] and fatty acids [6, 7] and the synthesis of phenylacetyl-
glutamine and hippurate [8].

Two procedures have been described for the preparation of aminoacyl adenyl-
ates. One of these [9] involves the condensation of the acid chloride of an
amino acid with silver adenylate. DeMoss et al. [9] obtained leucyl adenylate
in 9 per cent yield by this procedure. The other method involves condensa-
tion of the free amino acid and adenylic acid in the presence of N,N'-dicyclo-
hexylcarbodiimide, and precipitation of the product by addition of acetone [2].
In our hands, these methods suffered from shortcomings often encountered in
the attempted synthesis of highly reactive molecules; thus, we obtained very
low yields of products of very low purity, and experienced great difficulty in
attempts at purification of the anhydrides.

In an effort to solve these problems, we investigated a number of synthetic
approaches, two of which have proved successful. The first consisted of con-
densing an N-carbobenzoxyamino acid anhydride with adenylic acid in aque-
ous pyridine; the N-carbobenzoxyaminoacyl adenylate was isolated, and the
blocking group was removed by catalytic hydrogenation with palladium [10].

Subsequently, an alternative synthesis was developed which has proved to be
more convenient; it will therefore be described here in greater detail. Equi-
molar quantities of N-carbobenzoxyamino acid and adenylic acid were shaken
in aqueous pyridine with an excess of N,N'-dicyclohexylcarbodiimide for sev-
eral hours. N,N'-Dicyclohexylurea was removed by filtration, and the filtrate
was treated with acetone to precipitate the product. Treatment with acetone
removed unreacted N-carbobenzoxyamino acid, N,N'-dicyclohexylcarbodi-
imide, and most of the pyridine and water. The precipitate was extracted
with ethylene glycol monomethyl ether; the product is soluble, and adenylic
acid is insoluble in this solvent. The N-carbobenzoxyaminoacyl adenylate was
precipitated from the extract by addition of ether. After catalytic hydrogena-
tion of the carbobenzoxy compound and removal of the catalyst, the super-
natant solution was lyophilized. The free aminoacyl adenylate was obtained
as a white powder.

The yields varied from 40 to 80 per cent; for example, glycyl-l-C14-adenylate
was obtained in 75 per cent yield, and the yield of DL-tryptophanyl-3-C14-adenyl-
ate was 44 per cent. The final products are estimated to be 70 to 80 per cent

CASTELFRANCO, MEISTER, AND MOLD AVE 117

pure on the basis of the hydroxamic acid-ferric chloride color reaction [11].
The major impurities consist of adenylic acid and amino acid formed by hy-
drolysis of the anhydride during hydrogenation. This method has been suc-
cessfully applied to the following amino acids: glycine, alanine, valine, leucine,
isoleucine, (3-alanine, proline, phenylalanine, tyrosine, tryptophan, glutamine,
asparagine, threonine, methionine, and serine. Studies on the remaining natural
amino acids are in progress.

Evidence for the proposed anhydride structure (fig. 1) includes the follow-
ing: (a) hydrolysis in alkaline solution yields equivalent quantities of adenylic
acid and amino acid; (b) reaction with hydroxylamine yields the correspond-
ing amino acid hydroxamates, which have been identified by paper chromatog-
raphy; and (c) paper ionophoretic study indicates that the aminoacyl adenyl-
ates have a net positive charge at pW 4.5. The possibility that the carboxyl
group of the amino acid may be linked to the adenylic acid moiety through
a group (e.g., 6-amino group of adenine) other than the phosphoric acid group
appears unlikely in view of the unusual reactivity of these compounds.3 An
additional property of a-aminoacyl adenylates which has proved of value in
characterization is their reactivity in the presence of the tryptophan-activating
enzyme and inorganic pyrophosphate to yield adenosine triphosphate [3].

a-Aminoacyl adenylates are very labile in aqueous solution at values of pH
above 5.5. Thus, at pH 7.2 at 37° C, they exhibited half-lives of 5 to 10 minutes.
On the other hand, carbobenzoxyaminoacyl adenylates suffered only about 10
to 20 per cent hydrolysis in 2 hours at 37° at pH. 7.2. Acetyl adenylate and
benzoyl adenylate exhibit stability of approximately the same order as carbo-
benzoxyaminoacyl adenylates under these conditions.

Preparation of glycyl-C14-adenylate and tryptophanyl-C14-adenylate made it
possible to study incorporation of the respective amino acid moieties into pro-
teins in systems previously employed for studies of amino acid incorporation.
The enzyme preparation was obtained as described by Zamecnik and Keller
[12]; it consisted of the supernatant solution (containing microsomes) ob-
tained by centrifuging a 25 per cent rat liver homogenate at 12,000^. This
preparation catalyzed the incorporation of amino acids into microsomal pro-
teins in the presence of adenosine triphosphate and an adenosine triphosphate-

3 Additional evidence for the proposed structure has recently been obtained. Thus, we
have been able to convert carbobenzoxytryptophanyl adenylate with nitrous acid to the
corresponding inosinic acid derivative. The latter compound has also been prepared by
condensing inosinic acid with N-carbobenzoxytryptophan by the procedure described in the
text for anhydrides of adenylic acid.

Acylation of the hydroxyl groups of ribose appears to be excluded. Thus, carbobenzoxy-
aminoacyl adenylates consumed theoretical quantities of periodate and, after reaction with
periodate, reacted with hydroxylamine to give the corresponding carbobenzoxyamino acid
hydroxamates. Paper ionophoretic study of the carbobenzoxyaminoacyl adenylates in borate
and other buffers was also consistent with the presence of free ribose hydroxyl groups;
the mobility of these compounds (and of adenylic acid) was greater in borate buffer than
in tris(hydroxymethyl) aminomethane buffer at pH 9.1.

118 MICROSOMAL PARTICLES

generating system. Thus, in the system of Zamecnik and Keller [12], we
observed 57 and 81 cpm/mg protein, respectively, with glycine-1-C14 and trypto-
phan-3-C14. As is indicated in table 1, incubation of the enzyme preparation
with radioactive glycyl adenylate resulted in significant incorporation of isotope
into the protein subsequently isolated. When the anhydride was hydrolyzed
with alkali before study, significant incorporation was not observed. Further-
more, equimolar concentrations of radioactive glycine plus adenylic acid did
not lead to incorporation. It should be emphasized that the specific activities of
the C14-aminoacyl adenylates were 5 per cent of the values for the free amino
acids used by us in the system of Zamecnik and Keller.

TABLE 1. Incorporation Studies

Reaction Mixtures * cpm/mg

Enzyme + Glycine-1-C14 + Adenylate 0.1 7

Enzyme + Glycyl-l-C14-adenylate 17.1

Enzyme t + Glycyl-l-C14-adenylate 195.

Enzyme t + Glycine-1-C14 + Adenylate 1 .3 1

* The reaction mixtures contained enzyme ( 1 ml)
and glycyl-C14-adenylate (2.5 /zmoles; 3.6 X 105 cpm)
in a final volume of 2.5 ml; incubated at 38° for
30 minutes. Similar results were obtained when
concentrations of aminoacyl adenylate from 10"2 M
to 10~6M were employed.

f Enzyme heated at 100° for 10 minutes.

Although these results appeared to be consistent with the hypothesis that
aminoacyl adenylates are intermediates in the incorporation of amino acids into
proteins, further experiments have raised the possibility that such incorpora-
tion may be explained in terms of nonenzymatic acylation of protein. Thus,
it was found that, when the enzyme preparation was heated for 10 minutes at
100° before incubation with C14-aminoacyl adenylate, the incorporation of
isotope into protein was considerably greater than with the unheated enzyme
preparation. In the experiments with heated enzyme, appreciable incorporation
of isotope did not occur with hydrolyzed anhydride preparations. Similar re-
sults have been obtained with tryptophanyl-C14-adenylate.

With both heated and unheated enzyme preparations, the binding of the in-
corporated amino acid to protein was quite stable and could be released only
by the drastic acid hydrolysis required for the cleavage of peptide bonds. Thus,
with glycine-l-C14-labeled protein (heated and unheated), the quantity of free
amino acids and the percentage of isotope released as C1402 by ninhydrin in-
creased in parallel fashion during hydrolysis with 6 N HC1 at 105° over a
period of 16 hours. When the proteins labeled by incubation with glycyl-1-C14-
adenylate and with tryptophanyl-3-C1 '-adenylate were treated with l-fluoro-2,4-
dinitrobenzene, followed by acid hydrolysis, dinitrophenylamino acid prepara-
tions were obtained which contained more than 70 per cent of the radioactivity

CASTELFRANCO, MEISTER, AND MOLDAVE 119

originally incorporated into the protein. Similar results were obtained with
heated and unheated enzyme preparations.

We have also found that, when glycyl-l-C14-adenylate is incubated with puri-
fied rat-liver ribonucleic acid obtained by phenol extraction [13], considerable
radioactivity remains associated with the ribonucleic acid preparation after ex-
haustive dialysis and ethanol precipitation. Approximately 40 per cent of the
radioactivity of the ribonucleic acid preparation was alkali-labile. When such
ribonucleic acid preparations were incubated with unheated and heated pro-
tein preparations, significant quantities of radioactivity were found in the protein
subsequently isolated. Thus, incubation of 1 micromole of glycyl-l-C14-adenyl-
ate with 50 mg of liver ribonucleic acid in 1 ml of water for 30 minutes at
38° gave a ribonucleic acid preparation containing 3000 cpm (after dialysis
and precipitation). When 20 mg of this C14-ribonucleic acid (1500 cpm) was
incubated with enzyme preparation (table 1) for 30 minutes at 38°, approxi-
mately 100 cpm was associated with the protein subsequently isolated. Similar
results were obtained with heated enzyme preparation.

The observed reactions of the aminoacyl adenylates with proteins are consist-
ent with the reactivity expected of anhydrides of this type. A similar result might
occur when proteins are treated with radioactive acetic anhydride. The reaction
of the aminoacyl adenylates with the heated protein preparations would appear
to be a nonenzymatic acylation reaction involving the free reactive groups of
proteins. Heat denaturation of the protein would be expected to expose a greater
number of amino groups to the action of the acylating agents.4 The extent to
which the labeling observed with unheated proteins may be due to an enzy-
matic mechanism is not known. Although we believe that in the present studies
transfer of the amino acid moieties from aminoacyl adenylates to proteins oc-
curred largely by a nonenzymatic process, the possibility cannot be excluded
that some of this transfer is enzymatically catalyzed. Perhaps nonenzymatic
acylation of proteins may also take place to some extent when amino acids are
incubated with adenosine triphosphate, an adenosine triphosphate-generating
system, and a suitable enzyme preparation. The aminoacyl adenylates formed
in the activation reaction [3] might be expected to react in such a manner.
Thus, the high reactivity of aminoacyl adenylates with protein and ribonucleic
acid may explain at least some of the reported [4, 12] incorporation phenomena.
It must be emphasized that the concentrations of aminoacyl adenylates pre-
sumably formed in the amino acid incorporation systems previously studied
would be expected to be considerably lower than the concentrations of aminoacyl
adenylates we have used. Much lower concentrations of aminoacyl adenylates
than those used here must be employed to make a meaningful comparison
of the labeling in the two systems. Such studies will require aminoacyl adenyl-
ates of considerably higher specific radioactivity. Although the present in-
vestigations raise the possibility that incorporation in cell-free systems into

4 Porter [14] has reported that heat denaturation of several proteins increases the number
of e-amino groups of lysine that can react with acylating agents.

120 MICROSOMAL PARTICLES

microsomal protein may be at least to some extent nonenzymatic, it is quite
possible that physiological mechanisms exist for the controlled transfer of the
amino acid moieties of aminoacyl adenylates.

Note Added in Proof

Zioudrou, Fujii, and Fruton have recently described the synthesis of C14-
tyrosinyl adenylate and C14-glycyltyrosinyl adenylate by a procedure similar to
ours. They observed labeling of heated and unheated rat-liver mitochondria
by these compounds and by their N-carbobenzoxy derivatives. They have also
concluded that the labeling of the mitochondria is due to nonenzymatic acyla-
tion (personal communication from Dr. J. S. Fruton; Proc. Natl. Acad. Sci.
U. S., in press).

Further studies in our laboratory indicate that enzymatically synthesized
tryptophanyl adenylate can acylate microsomal preparations and also other pro-
teins (e.g., bovine serum albumin, ovalbumin) ; these experiments were carried
out with systems containing pancreatic tryptophan-activating enzyme, ATP,
magnesium ions, and acceptor protein. Labeling of ribonucleic acid prepara-
tions was also observed by such systems. The recent findings of Berg and
Ofengand [15] and of Schweet, Bovard, Allen, and Glassman [16] are consistent
with the possibility that specific binding sites for amino acids exist on soluble
ribonucleic acid molecules. Whether such specific binding of amino acids to
ribonucleic acid can be obtained with chemically synthesized aminoacyl adenyl-
ates remains to be determined. The present studies emphasize the importance
of isolating specific proteins in experiments on protein biosynthesis; the recent
report of Bates, Craddock, and Simpson [17] on the incorporation of valine into
mitochondrial cytochrome c appears to be a significant step in this direction.

REFERENCES

1. M. B. Hoagland, E. B. Keller, and G. D. Novelli, Proc. Natl. Acad. Sci. U. S.,
P. C. Zamecnik, /. Biol. Chem. 218, 345 42, 325 (1956).

(1956). 10. M. Bergmann and L. Zervas, Ber.,

2. P. Berg, Federation Proc, 16, 152 67, 1192 (1932).

(1957). 11. F. Lipmann and L. C. Turtle, /. Biol.

3. M. Karasek, P. Castelfranco, P. R. Chem., 159, 21 (1945).
Krishnaswamy, and A. Meister, paper 13 12. P. C. Zamecnik and E. G. Keller,
of this volume. /. Biol. Chem., 209, 337 (1954).

4. M. B. Hoagland, P. C. Zamecnik, and 13. A. Gierer and G. Schramm, Nature,
M. L. Stephenson, Biochim. et Biophys. 177, 702 (1956).

Acta, 24, 215 (1957). 14. R. R. Porter, Biochim. et Biophys.

5. P. Berg, /. Biol. Chem., 222, 1015 Acta, 2, 105 (1948).

(1956). 15. P. Berg and E. J. Ofengand, Proc.

6. W. P. Jencks and F. Lipmann, /. Biol. Natl. Acad. Sci. U. S.. 44, 78 (1958).
Chem., 225, 207 (1957). 16. R. S. Schweet, F. C. Bovard, E.

7. H. S. Moyed and F. Lipmann, /. Bac- Allen, and E. Glassman, Proc. Natl. Acad,
teriol, 73, 117 (1957). Sci. U. S., 44, 173 (1958).

8. K. Moldave and A. Meister, /. Biol. 17. H. M. Bates, V. M. Craddock, and
Chem., 229, 463 (1957). M. V. Simpson, /. Am. Chem. Soc, 80,

9. J. A. DeMoss, S. M. Genuth, and 1000 (1958).

The Synthesis of Hydroxyproline
within Osteoblasts

[Abstract]

SYLVIA FITTON JACKSON

Medical Research Council Biophysics Research Unit
W heatstone Laboratory , King's College, London

Biochemical and morphological methods are being used to study the stages
of synthesis of intercellular material in active collagen-producing tissue cultures.
The direct oxidation of proline already bound in peptide linkage may be an
important step in the sequence of the synthetic processes which lead to the
formation of collagen protein [Stetten, 1949]. In previous work it has been
found that appreciable amounts of protein-bound hydroxyproline were formed
during the first 24 hr of culture before the appearance of characteristic collagen
fibrils [Fitton Jackson and Smith, 1957]. Free C14-L-proline was also readily
incorporated into the proteins of the growing tissue, and as much as 20 per
cent was converted to protein-bound C14-hydroxyproline [Smith and Fitton
Jackson, 1957].

Cell fractionation studies have been made on similar tissue cultures in an
attempt to establish whether the site of incorporation of free proline into the
proteins of the cell was the same as that of the formation of the protein-bound
hydroxyproline. The cultures were grown in contact with C14-L-proline for
various times and subsequently homogenized and subjected to differential cen-
trifugation in 0.88 M sucrose solution. The amount of labeled proline incor-
porated and converted to hydroxyproline in the six isolated fractions was meas-
ured. Observations were made in parallel on the morphology of the whole
cells and the various cellular fractions by means of the electron microscope.

Chemical analyses demonstrated the consistent presence of protein-bound
hydroxyproline in the fractions of larger particle size (3000 A). The results

121

122 MICROSOMAL PARTICLES

also showed that, under the influence of the cells, free C1 l-L-proline was most
rapidly incorporated into the proteins of the supernatant (obtained after final
centrifugation), and into the "small-granule" fraction of the cytoplasm (sedi-
mented at 105,000^) ; these fractions also contained the greatest amount of
ribose. Subsequently part of the labeled proline appeared as hydroxyproline in
a large-granule fraction (~3000 A particle size) as well as in fractions of larger
particle size; for example, after 21 hours of culture growth followed by contact
with radioactive proline for 1 hr it was found that for every 100 residues of
protein-bound proline 16.9 residues were hydroxylated in this fraction. With
longer contact times the radioactivity of the larger-granule fractions increased
considerably.

The significance of these results in relation to the synthesis of collagen pro-
tein by the osteoblasts and in the mechanism of the formation of typical collagen
fibrils was discussed.

REFERENCES

M. R. Stetten, 1949, /. Biol Chew., 181, R. H. Smith and S. Fitton Jackson, 1957,

31. /. Biophys. Biochem. CytoL, 3, 692.

S. Fitton Jackson and R. H. Smith, 1957,
/. Biophys. Biochem. CytoL, 3, 679.

Studies on Amino Acid Incorporation in
Bacteria Using Ionizing Radiation

ELLIS KEMPNER ERNEST POLLARD

Biophysics Department, Yale University a

In recent years a growing body of evidence supports the idea that ribonucleo-
protein particles form at least one of the sites of protein synthesis [1]. Evidence
regarding this highly interesting system can be obtained by working with cell-
free systems of purified particles, and undoubtedly such evidence is valuable and
convincing. The drastic destruction of cell organization which is involved, how-
ever, leaves the question that perhaps the whole nature of amino acid incor-
poration is not being observed, but only the part that can survive the disrup-
tion of order in the cell. A method of study having the great advantage that
the cell is intact, or very nearly so, throughout the whole process is the use of
ionizing radiation as a powerful local disruptive agent. Such radiation is able
to penetrate all parts of the cell; it acts only at single, nearly isolated points, and
is wholly without action elsewhere. Such high-energy spot probes, or line
probes, can be employed against the organization of the cell, and, from the
effect on any particular part of the synthetic process, deductions can be made
regarding the process itself. Under good conditions, information can be ob-
tained on the following points: (a) the approximate size (within a factor of
2 in volume) of the region concerned with synthesis and sensitive to radiation;
(b) the approximate thickness (within a factor of 2 or 3), and hence the ap-
proximate length; and (c) the sensitivity of the synthetic region to radiation
(within a factor of 2) . These data can be compared with the sizes, thicknesses,
and lengths of cellular elements that could take part in the synthetic process.
This comparison can then be used as one more piece of evidence regarding the
nature of the process. A start along this line of investigation was made by

1 Aided by a grant from the John A. Hartford Foundation.

123

124 MICROSOMAL PARTICLES

Hutchinson, Morowitz, and Kempner [2]. If sources of radiation are available,
the method is, relatively speaking, technically easy, and therefore attractive. It
is necessary, however, to be aware of the uncertainties of interpretation, to be
sure that misleading deductions have not been made. The use of ionizing
radiation to study cellular processes has been under intensive study in this
laboratory for several years [2, 3, 4, 5, 6], and therefore a summary of the find-
ings seems worth while, so that the validity of the conclusions can be estimated.

The two major classes of biological macromolecules, proteins and nucleic
acids, appear to be very sensitive to ionizing radiation. An enzyme molecule
loses its activity if a cluster of ions forms anywhere inside the molecule; an
antigenic protein loses its ability to combine with antibody if such a cluster
forms in a volume somewhat smaller than that of the protein. DNA, as trans-
forming principle, loses its function if such a cluster forms within a unit of
about 300,000 molecular weight. If irradiations are carried out in solution, re-
action products can move around, and they may have marked inactivating
power. Studies by Hutchinson [7] on yeast cells in various conditions of mois-
ture indicate that in the cell such reaction products carry their effectiveness
over a distance of only 30 A. All these effects can be modified by factors of
about 2 by several environmental conditions, notably oxygen tension and degree
of aggregation between protein molecules. Thus, until the final sorting out of
cause and effect is accomplished, the statistical interpretation of radiation effects
must be considered to be approximate only. Even so, it is valuable as an aid
in studying an important, inaccessible process. To give some idea of the va-
lidity of the conclusions drawn we reproduce here a diagram, prepared by
W. R. Guild, showing the relation between the "target molecular weight" de-
rived from the statistical radiation analysis of radiation inactivation and the
accepted molecular weights. Since the diagram is a log-log plot, it should not
be viewed over-optimistically, but the reason can be seen for the claim that a
factor of 2 is normally all that is involved as error.

In order to gain the maximum information from irradiation studies, at least
three, and preferably more, types of irradiation should be carried out: (1) Irra-
diation by radiation sources very rich in fast electrons, as, for example, electrons
themselves, of energy 0.5 Mev or more, or y-ray sources of energy in excess of 1
Mev, where the secondary electrons due to Compton recoil and photoelectric ab-
sorption have energies, in the main, in excess of 0.5 Mev. (2) Irradiation by
heavy particles of variable rates of energy loss. Such particles have dense ioniza-
tion, largely confined to tracks, and they give a different distribution of local en-
ergy releases from fast electrons. Heavy particles of at least two energies should
be used, to give a range of separation of energy releases. In our experiments we
have employed cobalt 60 y radiation, deuterons of varied energies, and a par-
ticles as bombarding agents. The results show that the uptake of methionine
into the protein fraction (fraction insoluble in cold trichloroacetic acid) is re-
tained unless very heavy irradiations are employed, and the sensitive region fits
very well with a sphere of radius 130 A. For the uptake of proline into the

KEMPNER AND POLLARD

125

10'

1 1

I06

M 1/

1 y 1 1

MOLECULAR WEIGHT

—

tr
2.03

^r •

—

in2

/ 1

1 1

10*

I03 10 I03

MOLECULAR WEIGHT

10"

10'

Fig. 1. Comparison of accepted molecular weights and those determined by target
analysis. We wish to thank W. R. Guild of our laboratory for permission to use this
figure.

same fraction the same analysis cannot hold; instead, the best fit to the data
is found for a long, thin, sensitive volume of radius roughly 11 A and length
roughly 2.2 microns. Thus the methionine incorporation can very well be identi-
fied with a process taking place in a microsomal particle, usually estimated as
having a radius of 100 A [8], while the proline incorporation appears to impli-
cate a whole chain, probably of nucleic acid, and may mean that the incorpora-
tion of proline is dependent on the integrity of a system that binds together
several microsomal particles. The experiments forming the basis for these con-
clusions can now be described.

MATERIALS AND METHODS

Cultures of Escherichia coli B, maintained in this laboratory for a year, were
grown with aeration at 37° C in an inorganic salt medium ("Minimal C
medium" [9]) containing 5 g of glucose per liter. Aeration was stopped when
the bacteria reached a concentration of approximately 5 X 10s cells/ml as read
in a Bausch and Lomb spectrophotometer. This is about the middle of the

126 MICROSOMAL PARTICLES

logarithmic-growth phase in this medium. The cells had a generation time
of about 50 minutes.

Cobalt 60 irradiation: Twenty-milliliter samples of bacteria were sealed in
culture tubes and placed in a cobalt 60 source which delivered 380,000 r/hour.
The temperature in the source was approximately 30° C. After irradiation,
the tubes were placed unopened in a 37° C water bath, and allowed to rise to
that temperature.

Cyclotron bombardment : Bacterial cells were spun down in a Sorvall Model
SS-1 centrifuge, and the pellet was resuspended at a concentration of 1 X 1010
cells/ml in minimal medium with no glucose added. One-tenth milliliter of
this suspension was placed on fine-pore filters (Millipore Filter Corporation)
and kept moist with a coarse filter backing containing distilled water. Irradia-
tion was performed in air at 0° C [10]. After irradiation, the bacteria were
resuspended in 10 ml of minimal medium with no glucose and allowed to
come to 37° C.

After irradiation and temperature equilibration, the bacterial suspension was
added to an equal volume of minimal medium containing glucose which was
aerated at 37° C. This incubation mixture contained 0.2 |jc of the radioisotope
to be studied. L-Methionine-S35, 5.5 mc/g, and L-cystine-S35, 14.5 mc/g, were
obtained from the Abbott Laboratories, Oak Ridge. L-Proline-C14, 8.9 mc/mM;
L-leucine-C14, 7.95 mc/mM; and D-glucose-C14, 2.06 Mc/mg, were supplied by
the Nuclear-Chicago Corporation. At various times during the incubation,
2-ml samples were taken for the "whole cell" and "TCA-insoluble" fractions
[2]. The filters on which these fractions were placed were then dried in air
and counted under a thin window (less than 150 ug/cm2) Geiger-Miiller coun-
ter. Background was about 17 counts per minute.

In order to provide the variety in distribution of ionization densities as men-
tioned in the introduction, cyclotron bombardments were carried out with vari-
ous thicknesses of aluminum absorber between the bacteria and the beam.
Since the beam has a definite range, the amount of inactivation it produced
varied with the absorbers, falling to zero when the range in absorber was ex-
ceeded, so that no deuterons hit the bacteria. From these curves the equiva-
lent absorption of each bacterial preparation could be measured and thus the
effective energy of the deuterons hitting them estimated. Since the density of
ionization varies with energy in a known way [5], the appropriate value for
each bombardment can be determined.

RESULTS

The effect of irradiation with cobalt 60 on the uptake of L-methionine is
shown in figure 2. Three sets of curves are presented. The first, on the left,
applies to the uptake of unirradiated E. coli. The counts per minute are plotted
against time for two samplings: the whole cell, as represented by the upper
line, and the fraction insoluble in cold trichloroacetic acid (TCA). The differ-

KEMPNER AND POLLARD

o.) Unirradiated b.) 253,000 r

127

0 760,000 r

15 0 5 10 15 0

INCUBATION TIME

I5min

Fig. 2. The effect of y radiation on methionine incorporation in E. coli. Except where
otherwise marked in these figures, solid circles correspond to the label in the intact cell,
and crosses refer to the cold-TCA-insoluble fraction. The difference between these two
curves, the "pool," is seen to decrease with increasing dose of radiation. Background was
20 cpm and has been subtracted.

o) Unirradiated

b.) 3 1 7, 000 r

c.) 950,000 r

15 0 5 10

INCUBATION TIME

15 0

15 min

Fig. 3. The effect of y radiation on proline incorporation. The cold-TCA-soluble "pool"
rises in magnitude and then falls off with dose.

128

MICROSOMAL PARTICLES

ence between these two, following Britten, Roberts, and French [11] and Cohen
and Rickenberg [12], is designated as the metabolic pool. In figure 2b, the
same process is employed on bacteria that have received 253,000 roentgens of
cobalt 60 irradiation. It can be seen that the total amount of radioactivity in-
corporated in the TCA-insoluble fraction is reduced; the rate of incorporation
is also less. The amount taken up by the whole cells is less, though not quite
to the same extent as the reduction in the TCA-insoluble fraction. In figure 2c,
still higher irradiation was employed, and the reductions in both fractions are
still more apparent.

Similar data are shown in figure 3 for the uptake of proline. There is a
marked difference from the effects with methionine in that more irradiation is
necessary to reduce the amount incorporated into any fraction, about twice the
dose in the TCA-insoluble fraction and nearly 10 times in the whole-cell frac-
tion. Because of the disparity in these effects it can be seen that the pool actu-
ally rises after bombardment.

For these two amino acids, with the exception of the proline pool, the amount
of activity remaining seems to be a diminishing exponential function of the
dose. The data scatter somewhat, but we have no real evidence in favor of a
multiple-hit type of process, where the activity remains nearly constant and
then rapidly falls. The per cent remaining activity for methionine incorpora-
tion is plotted against dose in figure 4. For proline incorporation the effect
of radiation is definitely less in the TCA-insoluble fraction than for methionine,
and very markedly less in the whole-cell case. Such dose effect curves can be
analyzed statistically in terms of an inactivation volume V, which is the sensi-
tive region that must escape an ionization in order to retain the effect being
measured. If the ionizations occur at a number / per unit volume, then the
average number of ionizations occurring in the sensitive region is IV, and by
the Poisson relation the probability that the region will escape is e~Iv. Thus
the natural logarithm of the ratio remaining to that in the unirradiated con-
trol should be —IV. For 37 per cent remaining, the value of IV is unity.

Table 1 summarizes the results of cobalt 60 studies. The first column gives
the 37 per cent dose found from the survival curves. The second column gives
the corresponding number of primary ionizations per cubic centimeter for such
a dose. Column three, the sensitive volume, is the reciprocal of the value in

TABLE 1. Summary of Incorporation Studies in the TCA-insoluble Fraction of E. coli

Irradiated with Cobalt 60 y Rays

Primary

Sensitive

Equivalent

37%

Ionizations

Volume,

Spherical

Dose, r

per cm3

cm3

Radius, A

Leucine C14

0.36X106

1.8 XlO17

5.6X10-18

110

Cystine S35

0.45x10°

2.3 XlO17

4.3 XlO-18

100

Methionine S35

0.20 X106

l.OxlO17

10.0 XlO"18

130

Proline C14

0.45x10°

2.3 XlO17

4.3 XlO"18

100

Glucose C14

0.84x10°

4.3 XlO17

2.3 XlO-18

KEMPNER AND POLLARD

129

1000

300

100

^ 30
\

3
O

° 10

Whole Cell

TCA
Insoluble

0.2 0.4 0.6 0.8

DOSE (ROENTGENS)

1.0

1.2x10

Fig. 4. Survival of methionine incorporation with Y-ray dose. The ordinate is on a
logarithmic scale. Pool fraction shows similar survival.

the second column. The last column, the "equivalent spherical radius," is the
value calculated assuming that the sensitive volume is the shape of a sphere;
it is listed for comparative purposes only.

Cyclotron irradiations produced similar effects. The data scatter even more,
probably owing to the difficulty in securing irradiations involving the same
time of exposure on the Millipore filter. There is quite clear evidence of a
reduction in activity, as can be seen from figure 5, where the uptake of methi-
onine is shown for control and two irradiated points. Uptake curves of the
sulfur-labeled amino acids do not extrapolate to zero counts per minute at zero
time, owing to adsorption of the label, and this correction is deducted in plot-
ting survival curves. Figure 6 shows analogous curves for proline incorpora-
tion, also after deuteron irradiation. These experiments were performed with-
out added aluminum absorber in the cyclotron beam.

Similar studies were carried out with various absorbing foils, and, from the
uptake curves, curves relating the per cent uptake remaining to the number of
deuterons per square centimeter used to irradiate were drawn for each foil
thickness. In each case, the 37 per cent dose (in deuterons per square centi-

130

MICROSOMAL PARTICLES

oJ Unirrodiated

1200

800-

400-

1200

10 2 10 2

b.) 11.3 x 10 deuterons/cm c.) 33 x 10 deuterons / cm

METHIONINE

600

400

200

6 0 5 10

INCUBATION TIME

15 0

15 min.

Fig. 5. The effect of deuteron bombardment on methionine incorporation. Count rate
at zero time is due to adsorption of the label.

1200"

800

400-

//r

a.) Unirrac /•'/ b.) 8.5 x lo'° deuterons/cm2 c.) 23.3 x I010 deuterons /cm2

•300 ^ _ 300r

10 15 0 5 10 15 0

INCUBATION TIME

10 15 min.

Fig. 6. The effect of deuteron bombardment on proline incorporation. Here the pool
decreases in absolute magnitude with dose.

KEMPNER AND POLLARD

131

meter) was found. The statistical analysis is similar. If we assume that a
deuteron is able to inactivate as long as it passes through the sensitive region,
then the area of the sensitive region is all that matters. On that basis, if D is
the number of deuterons per square centimeter and S is the area of the sensi-
tive unit concerned with amino acid uptake, we see that the average number
of deuterons per target is SD, and once again the probability of escape is e~SD .
Therefore at the value of D for 37 per cent remaining, SD = \. S is referred to
as the "cross section." It must be remembered that a deuteron may not be per-
fectly efficient in producing inactivation, and in particular, if the ionizations
have a finite separation, then a thin target can be "straddled." This effect is
apparently present for proline uptake.

In figure 7, the cross section is plotted against the air equivalent of the foil
thickness for the TCA-insoluble fraction containing C14 proline. The maxi-
mum of this curve is near 26 X 10"12 cm2 at a value of 5.5 cm air equivalent of
foil. From the residual range of the beam, the value of the linear energy trans-
fer (LET) at this cross section is found to be 400 electron volts per 100 A.

1 2

\-
o

to
o
or
o

10 -

- xl0"'2cm2

PROLINE

TCA INSOL.

•/^

•\

< i

A.
i i iV

•-!

16 cm.

ABSORPTION (AIR EQUIVALENT)

Fig. 7. Measured cross section for proline incorporation into the TCA-insoluble portion
of E. coli after deuteron bombardment as a function of added aluminum in the cyclotron
beam; 12.5 cm is seen to be the range of the deuteron beam in air.

Incorporation studies after a-particle bombardment are indicated in figure 8
for proline. The survival curves for such studies were reasonably exponential,
with a 37 per cent dose for the TCA-insoluble portion labeled with proline
of about 2.7 X 1010 a particles per cm2.

The results of y-ray, deuteron, and a-particle irradiations are combined in
the LET plot [5] in figure 9. The two relations, in terms of volume V and
area S, reduce to the same expression when the density of ionization, or linear
energy transfer, is low. Under such circumstances, if / is the number of pri-
mary ionizations per unit length, l — Di, so that we obtain for the probability
of escape e~DiV, or S=iV. Thus the value of S close to the origin can be found

132

MICROSOMAL PARTICLES

PROLINE

6000-

5000-

4000

3000

2000-

1000-

15 0 5 10 15 0 5 10 I5min

INCUBATION TIME

Fig. 8. The effect of a-particle irradiation on proline incorporation. The pool decreases
with increasing dose.

z
o

a so

CI4 Proline

TCA Insoluble Fraction

S35 Methionine

400 800 1200

LINEAR ENERGY TRANSFER

Jl-

1600 e.v./IOOA

Fig. 9. Measured cross sections for proline (crosses, dashed line) and methionine (solid
circles, solid line) incorporation into the cold-TCA-insoluble fraction as a function of the
rate of energy loss (LET). Slopes at the origin are calculated from y-ray experiments as
explained in the text.

by substituting the product of i and V as already found from irradiation with
Y rays. Such a relation only applies close to the origin, but can be used to de-
termine the initial slope. To a reasonable approximation, i is the energy loss
per centimeter, in electron volts, divided by 110.

KEMPNER AND POLLARD 133

The slopes at the origin (dashed for proline, solid for methionine) are cal-
culated from the sensitive volumes found with y radiation. The points near
400 ev/100 A are from the deuteron experiments as described above. The LET
for a particles in our experimental arrangement was 1500 ev/100 A, and the
cross sections found by this method are shown at that LET value.

DISCUSSION

The analysis of the methionine-cold-TCA-insoluble fraction data indicates a
spherical target. The radius calculated from the inactivation volume is 130 A;
the radius calculated from the cross-sectional area is 127 A. The agreement is
well within the uncertainties of this method of study.

The proline data are somewhat more complex. The upward concavity near
the origin indicates a rather complex response to radiation. It can be explained
most simply by supposing that closely grouped ionizations are more effective
than single ionizations, and the rapid change of slope suggests that this multi-
plicity is small, probably a double ionization requirement [5]. There is no
agreement between the area and volume determinations if a spherical target
is assumed, no matter which ionization requirement (one, two, three, or four)
is used. The only simple model that fits the data is a long, thin rod [10]. For
a double ionization requirement, the volume and area determinations lead to
a model of a rod of 11 A radius and 2.2 u length. The analysis based upon
the model of a thin plate leads to substantially the same result, an extremely
long plate of small cross-sectional area.

Preliminary experiments with deuteron-bombarded E. coli cells indicate that
the incorporation of glucose into the TCA-insoluble portion of these cells re-
quires the integrity of a sphere of approximately 80 A radius. Since, in these
experiments, glucose was the single carbon source available to the organisms,
it seems reasonable to conclude that amino acids are produced in the cells in
spheres of this size. Since the doses required to stop glucose incorporation into
the cold-TCA-insoluble fraction are large compared with the doses required to
stop exogenous methionine from being incorporated into the same fraction, it
would appear that the formation of proteins from amino acids is not directly
related to the incorporation of glucose. Further work on this very interesting
result is clearly necessary.

If the two radiation-sensitive spheres found for glucose and methionine in-
corporation were assumed to have a density of 1.3, and to be sedimenting in a
solution of viscosity 1.5 centipoises, then from

d2 = lSv)sV/(l-Vp)

it can be calculated that the spheres associated with glucose incorporation
(160 A diameter) should have a sedimentation constant of the order of 25 S,
while the larger, methionine-associated sphere, 260 A in diameter, should have
a sedimentation constant of about 77 S. Since particles of such size have been

134 MICROSOMAL PARTICLES

reported in extracts of E. coli [13], our work can be interpreted as support for
the functional character of these particles.

If the incorporation of exogenous amino acids into a TCA-insoluble form
corresponds to protein synthesis, then the conclusion that at least two different
amino acids are incorporated by two different structures within the cell logically
leads to a model in which protein synthesis takes place in at least two steps.
If the two units found by radiation were simultaneously required and both
essential for protein synthesis, then methionine and proline would be expected
to give the same radiation targets. The fact that targets of quite different char-
acter are found indicates that there must be some difference between the two.
It is clearly interesting to continue the studies to see whether groupings of
types of target exist.

Actually, that an ionization in a cellular unit which is probably composed
of a dozen or so subunits should destroy its function is surprising. It seems
certain that the effect of radiation on one part can precipitate a disruptive effect
on the whole. This suggestion has already been made by Billen and Volkin
[14]. Our data tend to support their conclusions.

ACKNOWLEDGMENTS

We wish to thank Messers P. Hanawalt, P. Schambra, and J. Lowry for
assistance in running the cyclotron. We are also grateful to Drs. R. Roberts,
D. Cowie, R. Britten, and E. Bolton for advice in rapid filtration procedures
as well as for many stimulating discussions.

SUMMARY

Ionizing radiations of different character, y rays, deuterons, and a particles,
were used to determine radiation targets for the incorporation of methionine,
proline, and glucose into the cold-TCA-insoluble fraction of Escherichia coli.
Spherical targets were found for methionine (260 A diameter) and glucose
(160 A diameter) incorporation. The target for proline incorporation is a long,
thin rod, 22 A in diameter and 2.2 u long. That the units associated with
methionine and glucose correspond closely in size to microsomal particles found
in cell debris is therefore evidence for the functional importance of these
structures.

The size and shape of the proline-associated incorporation target appear to
be those of a nucleic acid unit, although it is not possible to distinguish be-
tween RNA and DNA by this method.

The fact that the target determined for glucose incorporation is smaller than
that found for the incorporation of the exogenous amino acid methionine indi-
cates that glucose is not directly connected with the binding of incorporated
amino acids into bacterial protein.

KEMPNER AND POLLARD

135

REFERENCES

LP. C. Zamecnik and E. B. Keller,
/. Biol. Chem., 209, 337 (1954); J. W.
Littlefield, E. B. Keller, J. Gross, and P. C.
Zamecnik, /. Biol. Chem., 217, 111 (1955).

2. F. Hutchinson, H. Morowitz, and E.
Kempner, Science, 126, 310 (1957).

3. E. C. Pollard, Advances in Biol, and
Med. Phys.,3, 153 (1953).

4. Conference on Ionizing Radiation
and the Cell, L. F. Nims, Chairman, Ann.
N. Y. Acad. Sci., 59, 467-664 (1955).

5. E. C. Pollard, W. R. Guild, F. Hutch-
inson, and R. B. Setlow, Progr. in Biophys.
and Biophys. Chem., 5, 72 (1955).

6. W. R. Guild and F. DeFilippes, Bio-
chim. et Biophys. Acta, 26, 241 (1957).

7. F. Hutchinson, Radiation Research, 7 ,
473 (1957).

8. H. K. Schachman, A. B. Pardee, and
R. Y. Stanier, Arch. Biochem. Biophys., 38,
245 (1952).

9. R. B. Roberts, D. B. Cowie, P. H.
Abelson, E. T. Bolton, and R. J. Britten,
Studies of Biosynthesis in Escherichia coli,
Carnegie Inst. Wash. Publ. 607, Washing-
ton, D. C, 1955.

10. E. C. Pollard, J. Setlow, and E.
Watts, Radiation Research, 8, 77 (1958).

11. R. J. Britten, R. B. Roberts, and E. F.
French, Proc. Natl. Acad. Sci. U. S., 41,
863 (1955).

12. G. N. Cohen and H. C. Rickenberg,
Compt. rend., 240, 2086 (1955).

13. Other papers this volume.

14. D. Billen and E. Volkin, /. Bac-
teriol, 67, 191 (1954).

The Effect of X Rays on the Incorporation
of Phosphorus and Sulfur into Escherichia colt

ERNEST POLLARD JANE KENNEDY

Biophysics Department^ Yale University

In the previous paper of this volume an account was given of preliminary
studies on the effect of ionizing radiation on the incorporation of amino acids
and glucose into a fraction of the bacterial cell that is not soluble in trichloro-
acetic acid. It is hoped that a continuation of such probing into the synthetic
processes in the cell by radiation will give some information on the nature of
the processes themselves. Though the subject of keenest interest at the moment
is undoubtedly the fate of an individual metabolite, and the way in which it
becomes incorporated, it seemed to be important to know something of the
way in which radiation affects the uptake of two much more generally utilized
elements of the growth medium, phosphate and sulfate. The studies to be de-
scribed were originally meant simply as a means of gaining general informa-
tion about the effect of radiation on the cell metabolism, to ensure that no seri-
ous discrepancies existed between the findings with one amino acid and the
whole metabolic process of the cell. During the studies some results appeared
that seem to indicate rather remarkable radiation sensitivities and are, more-
over, of interest in themselves. For instance, the ability of the cell to incor-
porate phosphate is remarkably sensitive to X radiation; the sulfate-incorpora-
tion ability is also highly sensitive, though less so; and sulfide incorporation is
definitely still less sensitive. The hypothesis that suggests itself is that the
phosphorus incorporation is determined by relatively large and sensitive units,
and that when these are damaged by radiation there is a proportionally smaller
phosphorus uptake. For sulfur uptake the results are in accord with the idea
that there is a considerable synthetic chain which presents sensitivity at a num-

1 Aided by a grant from the United States Public Health Service.

136

POLLARD AND KENNEDY 137

ber of loci. The results are only preliminary, but they do contribute to an
emerging picture of cellular processes which is worth some consideration.

Closely related to this work is a series of studies by Billen and Volkin [1],
Billen and Lichstein [2], Billen, Stapleton, and Hollaender [3], and Billen [4]
on the effect of X rays on several factors in E. coli. Their results will be
discussed later. Some work by Labaw, Mosley, and Wyckoff [9] is also of in-
terest, though directed at bacteriophage development.

PRINCIPLES OF RADIATION INACTIVATION STUDIES

For these experiments the simplest and most available source of ionizing
radiation, X rays, was used. X rays produce ionizations which are distributed
along the tracks of secondary electrons, light particles that ionize relatively
sparsely. In our arrangements, where 250-kv X rays filtered through 1 mm of
aluminum were used, the average secondary-electron energy is in the neigh-
borhood of 50 kv, and so produces ionizations which are generally separated
by distances of over 1000 A. Since the electrons scatter readily, the net effect
is to produce local energy releases, which average 110 ev each, at random
throughout the bacterium. The number per unit volume depends on the dose
of radiation, and for the purposes of this paper will be taken as 5 X lO^/cc/r,
which assumes a density of a little over unity for the bacterium.

Many previous studies, quoted in the preceding paper of this volume, have
shown that the energy releases can remove biological potency from quite large
biological molecules. In proteins it is thought that the reason is the migration
of the positive charge left by ionization, along a covalently bonded structure
until it reaches a weak point, where chemical action by water or oxygen can
cause a destructive chemical change. Then ionization anywhere in the cova-
lently bonded structure is destructive. For nucleic acids the best evidence sug-
gests that either a break in the chain can result, or a cross linking of two chains
can take place, both drastic events. In any event, the philosophy of these ex-
periments is to estimate the chance of complete escape from any ionization at
all, arguing that the chance of being ionized and escaping any effect is statisti-
cally too low to be worth considering. Such estimates are, of course, tentative
to some degree. Nevertheless, the sum of 10 years' work in our laboratory, to-
gether with the findings of many others, renders them plausible.

The structure that is being bombarded by these random energy releases is
shown in the section electron micrograph in figure 1. The section is of two
Escherichia coli bacteria, one of which has passed into a rather filamentous
form, and the other of which is normal. The sectioning and microscopy are
entirely due to L. Caro of our laboratory, and the illustration is presented by
his permission. It shows clearly the presence of an inner region, almost cer-
tainly the locale of the DNA, as has been shown by Caro, Van Tubergen, and
Forro [5], and an outer region which contains microsomal particles, or "ribo-
somes," and which is presumed to be the region of protein synthesis. Figure 2

138

MICROSOMAL PARTICLES

Fig. 1. An electron micrograph of a thin section of E. coli, taken by L. Caro. The
ribosome granules can be seen in the body of the cell. The inner region, presumed to
contain DNA, can be seen, and the structure of the cell wall is also apparent.

is a schematic drawing of the escape process, in which one of the ribosomes
is indicated as present in a region 1000 A square and 100 A deep, with the
appropriate energy releases accompanying it. On the left is the effect produced
by 10,000 r, and the microsomal particle is shown to scale with the single ioniza-
tion which is to be expected accompanying it. According to the studies of
Hutchinson [6], in a cell containing the normal proportion of water there can

POLLARD AND KENNEDY

\0,000r \00,000r

139

1,000,000

~b.000.000r

ClipS

98%

75%

0.07%

Fig. 2. A schematic representation of the statistical analysis of radiation damage. It is
supposed that to escape damage a microsomal particle must escape an ionization in its
structure, or even the diffusion of an active agent from ionization to its structure. The
probability of escape can be seen to be clearly dependent on the particle size and the amount
of ionization.

be a migration by diffusion of the energy release to the extent of 30 A. This
little spread of energy has been indicated by a dotted line around the ioniza-
tion. It can be seen that the chance of escape at 10,000 r is very good, the cal-
culated figure being 98 per cent. In the second region the microsomal particle
has been given an increase of 30 A in radius to allow for the possibility of
migration of energy, and now each ionization has to be considered as a single
dot. The chance of escape is now less but still high; it is estimated at 75 per
cent. With 1,000,000 r it is only 8 per cent, and with 3,000,000 r the chance of
escape is very remote indeed, and it is expressed by the unlikely figure of 0.07
per cent.

The experimental method, therefore, is to expose bacterial cultures held in
a static condition by being chilled to 4° C to X rays, to permit them to warm
up, and then to add labeled phosphate or sulfate to them and observe the up-
take of radioactivity as compared with the uptake of the unirradiated prepara-
tion. The ratio of the two is then compared with the chance of escape, and
deductions regarding the size of the particle are made from this probability.

EXPERIMENTAL METHOD

The experiments with phosphate are complicated by the fact that very com-
monly phosphate buffer is used in minimal media. This means that a large
excess of cold phosphate is present, rendering uptake somewhat difficult.
Accordingly the procedure we evolved was to grow the bacteria overnight in
the C minimal medium as described by Roberts, Abelson, Bolton, Britten, and
Cowie [7] and then transfer for a 90-minute growth period to Tris buffered
medium containing 80 mg of phosphate/1. The 90 minutes permitted the bac-
teria to establish themselves satisfactorily in the new medium; they were
then chilled and irradiated with a 250-kv X-ray machine at approximately
3000 r/minute. Subsequent to irradiation both the control and the irradiated
sample were warmed in a 37° bath and an appropriate number of counts of
radioactive phosphate was added to each one. The bacteria were sampled at

140

MICROSOMAL PARTICLES

different intervals of time by means of a small syringe and filtered through an
S and S filter. The filters, after being stored to dry, were counted in the
ordinary way.

For sulfate the same procedure was adopted because it was thought worth
while to compare phosphate and sulfate uptake under similar conditions. For
the uptake of sulfides we encountered considerable difficulty, as was pointed
out by Cowie, in that the sulfide very readily formed sulfate and does not stay
in its original form. We overcame this to some extent, though not always con-
sistently, by having an excess of cold sulfide present and by working as rapidly
as was consistent with the experiment. When fresh radioactive sulfide and
fresh sodium sulfide were used the data indicated a very clear difference from
those obtained with sulfate and could also be made to give consistent results
though not quite to the same degree as sulfate uptake.

EXPERIMENTAL RESULTS

The form of an uptake curve for phosphate is shown in figure 3. The ex-
periments differ from those of the previous paper in that there is no limitation
in the amount of tracer and so the bacteria can continue to incorporate phos-
phorus throughout a whole period of 2 hours. It can be seen that for the con-
trol and the slightly irradiated points there is an exponential uptake of P32 but
that at the higher doses the uptake becomes linear. By comparing the early
slopes of the lines a survival ratio can be obtained which can then be plotted

2000--

I600-"

i-
z

1200 --

800- -

400" "

PHOSPHATE

CONTROL
/2,900r

20 40 60 80 100

TIME OF UPTAKE

I20MIN.

Fig. 3. The results of one individual experiment on phosphate uptake. The control and
least-irradiated cultures show exponential uptake; the more heavily irradiated cultures show
reduced linear uptake.

POLLARD AND KENNEDY

141

against the dose. The change from logarithmic to linear increase is significant
and probably means, as pointed out by Cohen [8], that some unit is being
formed in the irradiated cell which is not suitable for further growth. The
cell is then continuing to operate on its existing material and not making fresh.

With radioactive sulfate the curves shown in figure 4 were obtained. These
are the results of a single run, and they are very similar to the uptake curve
for phosphate except that the sensitivity to ionizing radiation is not quite so
great. In the course of study we noticed that the initial part of the curve did
not seem to be as greatly affected by ionizing radiation as later stages, and
accordingly a deliberate attempt to study this was made, with the results shown
in figure 5. Much higher doses were employed, and it is apparent that the
effect of quite high doses on sulfate uptake is to cause a breaking away from
an initial line which occurs progressively earlier and earlier in the uptake proc-
ess. The final slope of the uptake is diminished as found previously, but the
breaking-away point seems to come later and later with lower and lower dose.

This again seems to indicate that the effect of radiation is on some mecha-
nism in the cell that prevents future development of means for further growth.

2800

2400

2000

1600

t-
z

o
o

1200-

800

400

CONTROL

/ 5,300 r

X/ 43,000 r

60 80 100

TfME OF UPTAKE

120 WIN.

Fig. 4. The results of one uptake experiment on sulfate. For low irradiations the ex-
ponential character remains, but at higher doses the uptake becomes linear. The uptake is
less sensitive than that of P04.

142

MICROSOMAL PARTICLES

CONTR<

1500-

SULFATE

1000- -

i-
z
z>
o
o

500"

39,000 r

x„— — 78,000 r

_o Ill.OOOr

60 80 100

TIME OF UPTAKE

120

140 MIN.

Fig. 5. The initial uptake of sulfate at higher doses. For 10 minutes, even at a dose of
111,000 r, the uptake is normal. Thereafter, the rate of uptake breaks away from normal,
at earlier times for higher doses. The implication is that some synthesis can go on with
the original synthetic apparatus but that adequate provision for future synthesis is not made
in irradiated cells.

The initial processes in the cell seem to be capable of continuing, but they run
out and the damage to the cell then becomes apparent.

The incorporation of sulfide was studied in much the same way. The results
again look very similar to those found for sulfate except that still higher doses
were necessary to produce an effect, and also there was some indication that
the inactivation instead of being linear was curved. This might mean that
more than one process is involved in sulfide uptake.

By way of check experiments, the number of colonies produced after irradi-
ation was also studied and the effect of radiation on the optical density was
measured. The optical density is that after a standard period of growth, usu-
ally about 80 minutes.

The results of all these experiments can be seen in figure 6, where curves for
per cent remaining versus radiation dose in roentgens are plotted. It can be
seen that by far the most sensitive factor is the formation of colonies and that
this is followed, in order, by phosphate and sulfate uptake, optical density,
and sulfide uptake. Lastly, the effect on the methionine uptake is very small
indeed and can hardly be plotted on the graph. Analyzed in terms of the
probability of escape as mentioned earlier, the data are presented in table 1.

POLLARD AND KENNEDY
100

143

30-

METHIONINE

■ l

OPTICAL DENSITY

COLONIES

\
\ SULFATE

PHOSPHATE \

■~" 50 lOOxlOOOr

RAOIATION DOSE

Fig. 6. Logarithmic dose response plots for colony counts, optical density, and phos-
phate, sulfate, sulfide, and methionine uptakes. The relative sensitivities can be analyzed
in terms of sensitive volumes, which are given in table 1.

The most surprising result is the great sensitivity of phosphorus uptake. It
is difficult to see how the relatively simple processes involving phosphate can
be involved with such large target units. A repeat of the uptake, using C mini-
mal medium, has been made, with results still indicating a great sensitivity,
though perhaps not as great as in Tris. We are at a loss to explain the finding,
and rather than speculate about the control of phosphorus uptake by large units
of DNA we prefer to continue studies aimed at finding the nature of the dam-
aged products. It should be pointed out that, with all three tracers, phosphate,

144

MICROSOMAL PARTICLES

TABLE 1

Sensitive

Fraction

Process

37% Dose, r

Volume, cc

of Cell

Colony formation

5,000

4.X10-16

2.7X10-*

Optical density

Initial slope

8,500

2.4 xlO"16

1.5 XlO-4

Final slope

43,000

4.7 xl0-1T

3 XlO"5

Phosphate uptake

14,000

1.4xl0^1G

9 XlO"5

Sulfate uptake

23,000

8.7 XlO-17

6 XlO"5

Sulfide uptake

40,000

5xl0-17

3 XlO"5

Methionine uptake

200,000

io-iT

6.5 XlO"6

sulfate, and sulfide, the initial uptake is unaltered. This finding was reported
previously by Billen and his associates [1, 2, 3, 4], and we confirm it.

For the sulfate and sulfide data a simple explanation can be advanced. It
is best seen diagrammatically as in figure 7. We suppose that the synthesis of
protein is determined by the presence of intact units of the size stated in the
previous paper for methionine. However, the protein made can be essential
for the enzymatic reduction of sulfate or combination of reduced sulfur with
other elements to form intermediates, finally ending in methionine, cysteine, or
glutathione. Each enzyme is a protein and probably requires methionine or
cysteine, i.e., sulfur in the right form. The entire uptake of sulfur is thus sensi-
tive in three ways: first as final incorporation into protein, second as requiring
the availability of a suitable enzyme for a needed process, third in terms of the
synthesis of such enzymes. The sensitivity of methionine incorporation then
requires only the functioning of one ribosome. Sulfate will not be incorporated,
however, if enzymes are inactivated (experience shows this inactivation to be
relatively difficult, as each enzyme molecule is small), or if enzyme synthesis
is stopped, or if protein synthesis is stopped. Thus a much larger inactivation
volume for sulfate uptake can be predicted. From the ratio of the inactivation
volume for sulfate uptake, after the initial stage is over, to that for methionine
uptake, the number of steps involved can be estimated. From table 1 the steps
from sulfate to methionine are 8.7, and from sulfide to methionine 5. The
process is shown schematically in figure 7. We make no claim for correct inter-
mediary biochemistry. If the explanation we propose is right it could be checked
against known intermediary processes by means of competition techniques.
This check is planned for future work.

One question naturally asked concerns any change in the fractions in the
cell after irradiation. We studied the four fractions: alcohol-soluble, cold-TCA-
soluble, hot-TCA-soluble, and residue. The results are shown in table 2. We
found the relative proportion of S35 in the hot-TCA-soluble and residue frac-
tions to depend somewhat on the time and temperature of exposure to hot TCA,
and we do not regard the variations in the hot-TCA fraction as significant.
Possibly the fall in the alcohol-soluble fraction after heavy irradiation in sulfate

POLLARD AND KENNEDY

145

METH

sq,gso3

<\aCT. METH S03

— "—J METHIONINE

R2SH

R2SH^

v^r.sh

s2o3

' f X '

;V> ^>

Fig. 7. A schematic diagram to indicate the reason for the high sensitivity of sulfate
uptake. A series of reduction and combination steps, requiring enzymes (indicated as
rectangles), has to be complete before the activated amino acid can be incorporated. Each
enzyme is made by a microsomal particle vulnerable to radiation. Hence the sensitivity for
a nine-step process is nine times higher than for a single step.

medium is a real effect. Otherwise the major conclusion is that no change in
the broad distribution is observed.

That the initial uptake is unchanged, but reduced uptake occurs sooner
for more heavily irradiated cells, argues that a considerable amount of incor-
poration can go with the damaged synthetic apparatus, but that an unbalance
occurs as the cell develops. If the damaged ribosomes were making enzymati-

TABLE 2. Cell Fractionation under Various Conditions

Fraction

Alcohol-soluble (lipids, protein)

Cold-TCA-soluble (transient inter-
mediates, glutathione)

Hot-TCA-soluble (nucleic acid
hydrolysates)

Residue (protein)

Unirradi-

ated

50 kr

ated

300,000 r

Sulfide

Sulfate

5.5

146

MICROSOMAL PARTICLES

cally impotent protein, such would be the case. The linear rather than expo-
nential uptake in damaged cells tends to argue in favor of this hypothesis. We
intend to study specific cell fractions as a check.

ACKNOWLEDGMENTS

We wish to thank Stuart Hauser for assistance in the later stages, particularly
for the data for figure 5. Discussions with Ellis Kempner have been most
useful.

SUMMARY

The uptake of P32 phosphate and S35 sulfate and sulfide by E. coli as in-
fluenced by X rays has been studied. The initial uptake is not greatly changed
by doses up to 100,000 r, but the rate of uptake is radically changed at later
times. Expressing the ratio of the final rate of uptake to that of unirradiated
controls in terms of dose we find a logarithmic relation. Analysis in terms of
a sensitive volume gives large sensitivities as follows:

Colony formation (for comparison)

P04 uptake

S04 uptake

S uptake

Methionine uptake (for comparison)

4 X 10-16 cc

1.4 X10-16

9.5 xlO-17
5xl0-17

io-17

The surprisingly large sensitivity for phosphate may mean that intact DNA
controls phosphorus uptake. The figures for sulfate, sulfide, and methionine
can be explained in terms of a synthetic chain of microsomal particles involv-
ing nine steps from S04 to protein and five from sulfide to protein.

REFERENCES

1. D. Billen and E. Volkin, Effect of
X-rays on the macromolecular organiza-
tion of E. coli B/r, /. Barter iol, 67, 191—
197 (1954).

2. D. Billen and H. C. Lichstein, Effect
of X-radiation on the adaptive formation
of formic hydrogenlyase, /. BacterioL, 63,
553-555 (1952).

3. D. Billen, G. E. Stapleton, and A.
Hollaender, The effect of X-radiation on
the respiration of E. coli, f. BacterioL, 65,
131 (1953).

4. D. Billen, Modification of the release
of cellular constituents by irradiated E.
coli, Arch. Biochem. Biophys., 67, 333—
340 (1957).

5. L. Caro, R. T. Van Tubergen, and F.
Forro, Jr., Radioautography of sectioned

bacteria, Biophysical Society Program Ab-
stract R 12, 1958.

6. F. Hutchinson, The distance that a
radical formed by ionizing radiation can
diffuse in a yeast cell, Radiation Re-
search, 7, 473-483 (1957).

7. R. B. Roberts, P. H. Abelson, D. B.
Cowie, E. T. Bolton, and R. J. Britten,
Studies of Biosynthesis in Escherichia coli,
Carnegie Inst. Wash. Publ. 607, Washing-
ton, D. C, 1955.

8. G. N. Cohen, Synthase de proteines
anormales chez Escherichia coli K-12 cul-
tive en presence de L-valine, Ann. inst.
Pasteur, 94, 15-30 (1958).

9. L. W. Labaw, V. M. Mosley, and
R. W. Wyckoff, Development of bacterio-
phage in x-ray inactivated bacteria, /.
BacterioL, 65, 330-336 (1953).

Statistical Relations in the Amino Acid Order
of Escherichia coli Protein1

HAROLD J. MOROWITZ

Biophysics Department, Yale University

Since proteins consist largely of chains of amino acids, a formal analogy is
suggested between protein structure and written language, which consists of
chains of letters. The analogy breaks down when we come to consider func-
tion. For the function (meaning) of language is completely determined by the
sequence of letters, whereas the function of proteins depends on the secondary
structure (coiling) was well as on the sequence. (In poetry, secondary struc-
ture is extremely important.)

One characteristic of both proteins and language is the nonrandom frequency
of occurrence of letters. Thus if we examine a long passage written in English
we get a rank frequency distribution represented by figure 1, which also shows
the rank frequency distribution of amino acids in Escherichia coli protein. Fur-
ther examination of language reveals certain high-frequency pairs, triplets, and
higher groupings of letters [1, 2].

A further feature shows up on inspection of amino acid composition of pro-
tein. The nonrandom distribution of amino acids that is apparent in over-all
collections of proteins arises from a similar nonrandomness in individual pro-
teins. There are notable exceptions to these relations, however, particularly in
structural proteins like silk and collagen.

A further question suggests itself. In proteins, are there pairs, triplets, and
higher amino acid sequences that occur with unexpectedly high or low fre-
quencies ? That is, are there laws, similar to the laws of language, governing
the ordering of amino acids in peptide chains ? These statistical relations could
be the result of thermodynamic stability, of evolutionary selection, or of the

1 This research aided by a grant from the United States Public Health Service.

147

148

MICROSOMAL PARTICLES

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AMINO ACID IN COLI PROTEIN

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\

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LETTER IN ENGLISH

Fig. 1.

mode of synthesis of protein. Regardless of their cause, knowledge of them
would place severe restrictions on our theories of synthetic cellular processes.
In recent years a number of workers have tried to elucidate relations among
amino acids by examining experimentally available sequences and frequencies
[3]. These attempts have been thwarted by some serious difficulties. The
known sequences are selected by the small size and ease of purification of the
proteins involved. The proteins are thus often unrelated as to source or func-
tion. The failure, in this limited range, to find constraints should therefore not
be taken as the final word on the problem.

MOROWITZ 149

An alternative method of investigating the ordering relations is to give up
dealing with pure proteins and examine mixtures of related proteins in an effort
to elucidate statistical relations. Thus we may inquire into the frequency dis-
tribution of N-terminal amino acids, C-terminal amino acids, amino acids made
N terminal by tryptic digestion, or any other experimentally available cut. We
may look at the entire collection of proteins from a single cell type, or any
subgrouping of proteins definable by some experimental procedure. It must be
kept in mind that we are seeking general laws for given collections of proteins.

The experimental procedures are derivable from the techniques of sequence
analysis [4]. In particular, use is made of the fluorodinitrobenzene reaction and
the Edman degradation [5]. The application of these techniques must be modi-
fied, as we are dealing with a collection of proteins rather than a single protein.

Since detailed experimental protocols are being published elsewhere, we shall
only outline the method here [6]. E. coli cells are grown on a defined medium
and randomly labeled with a single radioactive amino acid (leucine) or in the
case of sulfur 35 label they are randomly labeled in three amino acids, cysteine,
cystine, and methionine. The cells are then washed, and extracted with ethanol,
ether, and hot trichloroacetic acid. The residual material containing most of
the cell protein and little else is subjected to oxidation with performic acid.
One portion of this material is subjected to fluorodinitrobenzene end group
analysis or conversion to the phenylthiohydantoin derivatives; a second portion
is digested to completion with trypsin and then subjected to the same end
group analysis. Quantitative end group analysis is carried out by counting the
spots on paper chromatograms that result from N-terminal amino acids and
interior amino acids.

We thus determine the frequency of occurrence of cysteine (including
cystine), methionine, and leucine in their positions, N terminal and following
the tryptic cut (which has a high degree of specificity for the carboxyl end of
arginine and lysine). The results of these experiments are shown in table 1.

TABLE 1

Percentage of
Amino Acid in

Percentage of
Amino Acid

N-Terminal

following

Amino Acid

Positions

Tryptic Cut

Methionine

3.6

13.4

Cystine and cysteine
Leucine

1.5
Less than 0.2

8.3
6-9

To interpret these results, let us first consider what results would have been
obtained if there were no ordering rules. That is, suppose that each protein
sequence was completely independent and any ordering was equally probable.
If there were a large number of different protein species, on a statistical basis
the amino acids would be expected to be randomly ordered. On the basis of

150 MICROSOMAL PARTICLES

average chain length as calculated from Van Slyke amino nitrogen determina-
tions any amino acid should be N terminal 1.5 per cent of the time and should
follow the tryptic cut about 14 per cent of the time.

Failure to observe these ratios indicates one of two situations: either there
are statistical rules of sequence, or a small number of protein species con-
tributes a large fraction of the total protein mass. Recent results reported on
the column separation of E. coli proteins [7] indicate that the protein mass
is distributed with reasonable uniformity among a large number of proteins or
protein groups. Thus, the results presented in table 1 suggest that statistical
rules of sequence are operative in specifying E. coli proteins.

REFERENCES

1. H. F. Gaines, Cryptanalysis, Dover 5. Fraenkel-Conrat et al., Methods of
Publications, Inc., 1956. Biochem. Anal, 2, 399 (1955).

2. R. B. Roberts et al., Studies of Bio- g. H. J. Morowitz and M. Spaulding,
synthesis in Escherichia coli, Carnegie Inst. Biochim. et Biophys. Acta, in press.
Wash Publ. 607, Washington, D. C, 1955 ? Work reported in ^ yolume by

3. G. Gamow et al., Advances in Biol. .

and Med. Phys., 4, 23 (1956). workers at Carnegie Institution of Wash-

4. C. B. Anfinsen and R. R. Redfield, ington, Department of Terrestrial Mag-
Advances in Protein Chem., 11, 1 (1956). netism.

The Formation of Protomorphs

FRANK T. McCLURE 1 RICHARD B. ROBERTS

Department of Terrestrial Magnetism
Carnegie Institution of Washington

Disrupted cells of Escherichia coli were suspended in a number of different
solutions to find which ones were suitable for making stable preparations of
ribosomes. Among those tested were some containing manganese and mag-
nesium, because these ions had been found essential for incorporation of amino
acids by cell-free systems [1, 2]. After standing for several hours these solu-
tions became turbid and finally gave a white precipitate of unusual appearance.

Examination of these solutions in the phase contrast microscope showed that
the turbidity was caused almost entirely by the presence of large numbers of
nearly spherical, highly refractile particles with diameters of 1 to 5 microns
(figs. 1 and 2).

The appearance of these cell-like particles in a solution that originally con-
tained nothing visible in the microscope was quite surprising. The formation
of large, stable aggregates with distinct boundaries from a fluid containing
macromolecules in a homogeneous suspension seemed to illustrate a process
which perhaps was important in the origin of life. Accordingly, we proceeded
to investigate some of their properties. It was soon found that the particles con-
tained protein, nucleic acid, and lipid in proportions typical of biological ma-
terials. Because they are formed from protoplasm and have distinctive shape
we refer to them as "protomorphs" to distinguish them from other particles
or structures that exist in the living cell.

The usual procedure for preparation of protomorphs is as follows: Harvest
10 g wet weight of Escherichia coli cells growing in synthetic medium "C" [3].
Wash twice with tris(hydroxymethyl)aminomethane-succinate buffer, 0.01 M,

1 Present address: Johns Hopkins University, Applied Physics Laboratory, 8121 Georgia
Avenue, Silver Spring, Maryland.

151

Fig. 1. Protomorphs in the solution from which they form.

Fig. 2. Aggregated protomorphs scraped off glass surface. Phase contrast photomicro-
graphs taken by W. R. Duryee.

152

McCLURE AND ROBERTS 153

pH 7.6 (TS). Suspend with 10 ml TS, and break the cells by forcing the sus-
pension through an orifice at 10,000 psi with a flow rate of roughly 2 ml/min.
Add TS to bring the total volume to 50 ml, and centrifuge 15 minutes at
40,000^. Discard the precipitate, which contains unbroken cells and large frag-
ments of cell walls. Centrifuge again at 105,000^ for 15 minutes, and discard
the precipitate, which contains smaller fragments of cell walls and a small pro-
portion of the ribosomes. Dilute the supernatant fluid with TS to 100 ml, and
add MgCL and M11CI2 to make it 0.005 M in each. In 5 to 48 hours the solu-
tion will show turbidity because of protomorph formation. The entire proce-
dure is carried out at 0° to 5 J C.

The yield of this procedure is variable, as is the time required for formation
of the protomorphs. Some of the sources of variability have been identified;
others remain unknown.

The addition of manganese is essential. No protomorphs were formed when
Mn++ was omitted even though adequate Mg++ was present. Higher concen-
trations of Mn" (0.01 M to 0.5 M) caused the formation of a precipitate of
particles of irregular shape and widely variable size. No attempt was made to
find other cations that might substitute for the Mn+\ The Mg++ was not essen-
tial but seemed to increase the yield. No difference was noted whether the
magnesium was added before or after the cells were broken; thus there would
appear to be no difference to protomorph formation whether the ribosomes
were in the 80 S form or not (see paper 3 of this volume).

No protomorphs were formed when the £>H of the solution was outside the
limits 7 to 8; a pH of 7.5 seemed to be optimum.

The concentration of orthophosphate affects the yield. When the cells are
carefully washed in TS before breaking, the phosphate of the growth medium
is removed and phosphate must be added to give a concentration of 10"3 M.
If higher quantities of phosphate are added the particles become less refractile
in appearance and are dark, rough, and "hairy." At still higher concentra-
tions of phosphate, precipitates are formed in manganese solutions lacking cel-
lular material. Although these inorganic precipitates have only a slight re-
semblance to the protomorphs, it is possible that the inorganic material provides
a framework on which the organic material deposits.

The concentration of the cellular material is important. When the usual pro-
cedure was followed a twofold dilution of the cell extract prevented the forma-
tion of protomorphs. The presence of cell-wall material was not important to
the yield; the yield was the same whether or not the centrifugation steps to re-
move cell walls and unbroken cells were omitted. When the wall material was
present the protomorphs appeared less smooth, as if irregular fragments of wall
had been incorporated.

The constituent responsible for the sensitivity to the concentration is prob-
ably deoxyribonucleic acid (DNA). The addition of DNAase invariably pre-
vents the formation of protomorphs. The pressure cell routinely used to dis-
rupt the cells also degrades DNA, as preparations of DNA lose their viscosity

154 MICROSOMAL PARTICLES

on being forced through the orifice of the pressure cell. Cell juices prepared
with the pressure cell do not show the peak characteristic of DNA in their
sedimentation diagram [4]. It is these preparations (made with the pressure
cell) that will not give protomorphs when diluted. In contrast, preparations
made by grinding the cells with alumina or by lysozyme treatment followed
by osmotic shock (methods which preserve DNA) do yield protomorphs even
at one-tenth the usual concentration. Finally, the addition of DNA to dilute
pressure-cell preparations restores the yield. It seems quite likely that varia-
tions in the pressure and in the conditions at the orifice during the disruption
of the cells affect the quantity of intact DNA remaining and thereby influence
the yield in an erratic way.

The formation of protomorphs was photographed by Drs. B. Hoyer and
N. Kramis, of the Rocky Mountain Laboratory, U. S. Public Health Service.
Their time-lapse photomicrography shows that the growth of an individual
protomorph from its first appearance to full size requires only a few minutes
after a much longer induction period. There may well be a slow process of
nucleation followed by a rapid process of growth. Neither fission nor fusion
played any part in the growth process.

Once formed, the protomorphs are stable. Unlike simple coacervate particles
which exist only in a narrow pYL range and have a strong tendency to fuse
or to dissolve, protomorphs can be handled like bacteria or yeast. There is no
difficulty in centrifuging the photomorphs into a pellet (lOOOg-) and resuspend-
ing in fresh media. They are quite stable in a number of ordinary media, and
persist for weeks even though overgrown by bacterial contamination. They
are not dissolved by short exposures to ammonia (1 M), 5 per cent trichloro-
acetic acid (TCA), ethanol, or ether. They are dissolved in 0.01 M ethylenedi-
aminetetraacetic acid to give a clear solution.

On standing, glass vessels containing protomorph suspensions develop a
white film over the surface. Microscopic examination of the material scraped
from the glass indicates that the protomorphs have formed a moderately well
packed monolayer on the surface (fig. 2).

The organic components of the particles had roughly the proportions found
in living tissues. They contain ultraviolet-absorbing material which hydrolyzes
to yield the bases expected from ribonucleic acid. In addition the diphenyl-
amine test [5] indicates that a small part (10 per cent) of the ultraviolet absorp-
tion is due to DNA. The ratio of nucleic acid to protein (measured by the
Folin reaction [6]) is 1/6 as compared to 1/4 in the bacterial juice. Paper
chromatography shows the presence of lipid material.

Incorporation experiments were carried out with thoroughly washed prepa-
rations of protomorphs. Radioactive phosphate was incorporated at a constant,
high rate for several hours. This process was not studied in any detail because
all the radioactivity so incorporated could be extracted with cold TCA and
there was no evidence of incorporation into macromolecules.

The incorporation of radioactive amino acids was of more interest. The re-

McCLURE AND ROBERTS

155

suits were erratic from one preparation to another, and the incorporation rate
was only 1/1000 that of intact cells at the highest. Accordingly contamination
by intact cells was a constant worry. The number of intact cells was estimated
both by plate counts and by microscopic examination. The uptake observed
was as much as 100 times that which could be attributed to the contaminants.

There were also several qualitative features which distinguish the behavior
of the protomorph preparation from that of intact cells. In the first place, the
incorporation doubled if ATP was added or if the concentration of the amino
acid mixture was doubled. These variations have little effect on incorporation
by whole cells. Secondly, the distribution of incorporated radioactivity among
the fractions soluble in cold TCA, alcohol, ether, and hot TCA was different
from that obtained with whole cells [3]. Finally, the hot-TCA-insoluble ma-
terial after hydrolysis yielded a pattern of radioactive amino acids different
from the mixture supplied and different from what would be incorporated by
whole cells.

Accordingly we believe that the observed incorporation was in fact real,
though not reproducible from day to day. Since these experiments were done
we have learned of the activity of the cell-wall fraction in protein synthesis
[7, 8]. In retrospect it seems quite likely that the variability in the synthetic
capacity of the protomorphs may have been due to a variability in their content
of cell-wall fragments.

These protomorphs are of course very different from the ribosomes which
are the subject of this symposium, but there may be a relationship between
them. It is a common belief that the bacterial cell is not a homogeneous mix-
ture of its various components; on the contrary, various lines of evidence indi-
cate that it has a high degree of organization. Organization in turn implies
the action of forces between the various constituents such as DNA, RNA, pro-
tein, and ribosomes. It is possible that the aggregation of these cellular con-
stituents into protomorphs may be another manifestation of the same forces
which maintain organization in the living cell and may furnish a material in
which the forces are more amenable to study. If so, the protomorphs may
eventually contribute to our knowledge of how the ribosomes are organized
within the cell.

REFERENCES

1. S. Spiegelman, A Symposium on the
Chemical Basis of Heredity, p. 232, Johns
Hopkins Press, 1957.

2. B. Nisman, personal communication,
1957.

3. R. B. Roberts, P. H. Abelson, D. B.
Cowie, E. T. Bolton, and R. J. Britten,
Studies of Biosynthesis in Escherichia coli,
Carnegie Inst. Wash. Publ. 607, 1955.

4. E. T. Bolton, B. H. Hoyer, and D. B.
Ritter, paper 3 of this volume.

5. Z. Dische, The Nucleic Acids, vol. I,
chapter 9, Academic Press, New York,
1955.

6. O. H. Lowry, N. J. Rosebrough, A. L.
Farr, and R. J. Randall, /. Biol. Chem.,
193, 265 (1951).

7. V. R. Srinivasan and S. Spiegelman,
Bacterial. Proc, 58, 101 (1958).

8. S. Ochoa, Spring Meeting, National
Academy of Sciences, 1958.

Structure of Microsomal Nucleoprotein
Particles from Pea Seedlings1

PAUL O. P. TS'O

Division of Biology, California Institute of Technology

Microsomal nucleoprotein particles from pea seedlings have been isolated
and characterized in our laboratories [1|. These particles have an RNA/pro-
tein ratio of 4/6 and a molecular weight of 4 to 4.5 X 10r>, and they appear in
the electron microscope as oblate spheroids (fig. 1). The microsomal nucleopro-
tein particles of pea seedlings thus seem to be similar to those of yeast [2], liver
[3], and perhaps also to those of bacteria as reported in this meeting.

The problem of the structure of the particles can be approached from at
least three lines of inquiry. (1) How are the smaller units of nucleoprotein put
together in the 80 S particles? (2) How are the RNA and protein put together
in the nucleoprotein? (3) If protein synthesis takes place in the particle, what
is the structural relation of the newly synthesized protein to the nucleoprotein
that constitutes the particle? The present paper is principally concerned with
the first of these questions.

Two salient features of the approach and of the interpretation should first be
noted. Experimentally, every precaution has been taken to assure that the
dissociation agents employed do not exert hydrolytic action on covalent bonds.
In interpreting the results of dissociation studies, it is assumed that all particles
in the preparation have similar gross structure. The simple ultracentrifugal
patterns of the dissociated particles, and the reversibility of certain dissociating
processes, seem to support this assumption. It is entirely conceivable, however,
that though in general features the subunits of all the particles may be very
similar, the cohesive strength with which they stick together inside the particle

1 This work was supported in part by grants Nos. Rg-3977 and Rg-5143 from the
National Institutes of Health, United States Public Health Service.

156

TS'O

157

Fig. 1. Electron micrograph of a freeze-dry preparation of microsomal particles from pea
seedlings. Magnification 41,000 X; thorium shadowed with an angle of 6.5:1.

may vary during the different phases of cell growth and differentiation. Pre-
liminary information suggesting that this is so has been reported in this meet-
ing: information concerning rapidly growing cells, such as those of yeast and
bacteria. Eventually, then, studies of structure of particles in the steady state
will evolve into studies of structure of particles through a life cycle.

Processes or agents that remove or substitute divalent ions from the particles
promote dissociation of the particles. For instance, in a 0.8 per cent particle
solution, in pH 6.5, 0.025 p, phosphate buffer, there is only 6 per cent dissocia-

158

MICROSOMAL PARTICLES

tion of 80 S particles into 60 S and 40 S units. In pH 7.5, 0.05 u, buffer, there
is 50 per cent dissociation, and in pW 8.5 buffer, there is over 80 per cent dis-
sociation (fig. 2a, b, c). Substantially, all the subunits in pH 7.5, 0.05 u, phos-
phate buffer can be returned to the form of 80 S particles either by addition of
magnesium (5 X 10"4 M) or by titration of the pH back to pH 6.5 (fig. 2d, e) .
Thus, the dissociation of 80 S particles to 60 S and 40 S units is a reversible
one. Increase in pH or concentration of phosphate buffer promotes the disso-
ciation by taking magnesium away from the particles. This interpretation is

PH:

(b)

7.5

(d)

(e)

Fig. 2. Ultracentrifuge patterns of particles in 0.05 fi K-phosphate buffer as affected by
pH. Speed 35,600 rpm. (a) In pH 6.5 buffer at 0° C for 13 hours. (/>) In pH 7.5 buffer
at 0° C for 16 hours. (<:) In pH 8.5 buffer at 0° C for l'/2 hours, (d) Solution in pH 7.5,
0° C for 2 hours, back-titrated with phosphoric acid to pH 6.5, stored at 0° C for 3 hours.
(e) Solution in pH 7.5, 0° C for V/z hours. MgCl2 added to final concentration of
5 X 10~4 M, stored at 0°C for 3 hours.

TS'O

159

further substantiated by experiments on dialysis of the particles. When a par-
ticle preparation is dialyzed against a buffer in which it is stable, dissociation
to 60 S and 40 S units occurs (fig. 5a, d). This dissociation is caused by re-
moval by dialysis of essential cofactor(s), since it can be prevented or reversed
by dialyzing the particles against the deproteinized supernatant (fig. 3>b, e).
The cofactor is present in the supernatant of the extract from which the par-
ticles were originally isolated; it is completely heat stable and not absorbed on
charcoal; finally, its protective and dissociation-reversing properties have been
duplicated by magnesium chloride, 5xl0"4 M (fig. 3c, /). Calcium chloride

Fig. 3. Ultracentrifuge patterns of particles after dialysis. Speed 36,500 rpm. (a) Dialy-
sis against 0.025 fi K-P04, pH 6.5, 2-4° C, for 13 hours, (b) Supernatant, plus 0.025 fi
K-P04, pH 65, 2-4° C, for 14 hours, (c) 0.025 /x K-P04, pH 6.5, 5 X 10"4 M MgCl2,
2-4° C, for 19 hours, (d) 0.025 ^ K-P04, pH 6.5, 2-4° C, for 24 hours, (e) Solution first
dialyzed in phosphate buffer to produce solution of (a), and the solution of (a) redialyzed
against supernatant with 0.025 p K-P04, pH 6.5, 2-4° C, 14 hours. (/) Solution first
dialyzed in phosphate buffer to produce a solution similar to (a), and the solution of 75,
60, and 40 S particles redialyzed against 0.025 p. K-P04, pH 6.5, with 5 X 10'4 M MgCl.,,
2-4° C, for 14 hours.

160

MICROSOMAL PARTICLES

at the same concentration has also been found to have protective properties.
Similar results have been obtained for yeast particles [4].

The dissociation of particles to smaller subunits is a relatively rapid process.
Thus, essentially the same proportions between 80, 60, and 40 S components
are obtained in runs on materials incubated at 0° C in phosphate buffer, pH
7.5, 0.05 |j, for 2 minutes or for 16 hours. No evidence was found in these ex-
periments to suggest that the 60 and 40 S components are formed by aggre-
gation of smaller products.

In concentrated potassium chloride solution (0.35 to 0.7 M), the particles
also dissociate into 60 and 40 S (fig. Ab, e) subunits, and this process too is sub-
stantially reversible (fig. 4&, c). Addition of magnesium chloride (0.015 to

KCI 0.7M

(a)

(b)

(c)

Nyy

(d)

(e)

Fig. 4. Ultracentrifuge patterns of particles in KCI solutions, (a) In 0.7 M KCI, 24° C,
for 30 minutes. Rotor speed 35,600 rpm. (/>) In 0.7 M KCI, 0° C, for 1 hour. Centrifuged
at 7° C. Rotor speed 42,040 rpm. (c) Solution of (b) dialyzed in 0.025 fi K-P()4, pH 6.5,
MgChl X 10"3 M, 2-4° C, for 14 hours. Centrifuged at 6.8° C. Rotor speed 42,040 rpm.
(d) In 0.7 M KCI with 0.015 M MgCh, 0° C, for 1 hour. Rotor speed 35,600 rpm. (<?) In
0.35 M KCI, 0° C, for 1 hour. Rotor speed 35,600 rpm.

TS'O

161

0.1 M) partly suppresses but does not completely eliminate the dissociation (fig.
4d). The effects of potassium chloride are, therefore, probably twofold. Potas-
sium ions appear to replace magnesium within the particle. The further effect
of potassium which cannot be suppressed by addition of magnesium is probably
due to ionic effects upon charged groups. An additional complication sets in
when the particles are exposed to potassium chloride solution at room tem-
perature. As will be described in more detail later, the RNA in the dissociated
particles is now hydrolyzed by the contaminating RNAase in the preparation
with a rate eight times higher than the rate constant for attack of the RNA
ot the nondissociated particles. The effect of the contaminating RNAase on
the particles in 0.7 M KCl can be observed by comparing figures 4a and \b.
There is much more material of very low sedimentation coefficient in the
room-temperature runs. Furthermore, the area ratio of the 80 S : 60 S : 40 S
components in the low-temperature run is 1.3 : 1.8 : 1.0, whereas in the run af-
fected by RNAase it is about 1:1:3. The 40 S component, therefore, may be
more resistant to RNAase than the others.

Magnesium ions can be further removed by EDTA. At neutral pH, the sys-
tem treated with EDTA aggregates at room temperature. Thus, this system
can be analyzed only at low temperature or at alkaline pH. In pH 6.5 phos-
phate buffer, and in the presence of 2.5 X 10~2 M EDTA, the particles disso-
ciate to yield components of 40 S (64 per cent), 26 S (30 per cent) and 3 to 6 S
(6 per cent) (fig. 5b). Lower concentrations of versene, such as 5 X 10"3 M,
yielded poor resolution of the 40 and 26 S components (fig. 5a). At higher
pH (9.0), higher concentrations of EDTA (5x10"' M), and room tempera-
ture, the particles dissociate into the 40 and 26 S units together with increased
amounts of material of 3 to 6 S(fig. 5c). Dialysis to remove EDTA and to
replenish magnesium causes aggregation of the system. It should be noted

(c)

Fig. 5. Ultracentrifuge patterns of particles in EDTA. Speed 42,040 rpm. (a) In
5 X 10-3 M EDTA, pH 6.6, 0.025 [x, K-P04 0° C, for 1 hour. Centrifuged at 4.6-6.0° C.
(b) In 2.5 X 10"2 M EDTA, pH 6.6, 0.025 ^ K-P04, 0° C, for 1 hour. Centrifuged at
6° C. (c) In 5 X 10"2 M EDTA, pH 9.0, 0.02 M K-COs, 0° C, for 3 hours. Centrifuged
at 20°C.

162

MICROSOMAL PARTICLES

that in this concentration of versene (0.05 M), and at pH 9.0, less than 1 per
cent of the original magnesium should remain with the particles.

The RNA/protein ratios of the dissociated components have been investi-
gated. The 60 and 40 S components were shown to be attacked and precipi-
tated by RNAase and protamine, indicating that they contain RNA. The
problem was then approached more quantitatively by comparing the ultracen-
trifuge patterns obtained by schlieren optics with those obtained by ultraviolet
absorption optics which measures the sedimentation of nucleic acid. It should
be noted that the concentration of particles in solutions employed for ultra-
violet absorption optics is 30 to 50 times lower than that used for schlieren optics.
In dilute solutions, the sedimentation coefficient will be higher and the reversi-
ble dissociation reaction will proceed further toward completion. Figure 6a
shows that all the nucleic acid moves as one component of 79 S in water. At
pH 7.5, 0.05 u, phosphate (fig. 6b), the particles are dissociated into two ultra-
violet-absorbing components of 37 and 59 S, those shown above to appear also
in the schlieren pattern. The absence of the 80 S particles, which do appear in
the schlieren pattern, in the ultraviolet absorption pattern in this phosphate
buffer is to be attributed to the complete dissociation of the original particles

c
o

Fig. 6. Ultracentrifuge ultraviolet absorption patterns of particles under dissociating and
nondissociating conditions, (a) In water. Rotor speed 25, 980 rpm. (b) In 0.05 /x K-P04,
pH 7.5, 0° C, for 1 hour. Centrifuged at 7.7° C. Rotor speed 37,020 rpm. (c) In 0.025 M
ETDA, pH 6.6, 0.025 p, K-P04, 0° C, for 1 hour. Centrifuged at 6.2° C. Rotor speed
42,040 rpm. (d) In 0.7 M KC1, 0° C, for 1 hour. Centrifuged at 8.5° C. Rotor speed
37,020 rpm.

TS'O 163

into the 59 and 39 S components at the low concentration. The ratio of the
amounts of ultraviolet-absorbing material, i.e., nucleic acid, contained in the
59 and 39 S boundaries is similar to the ratio of amounts of material contained
in the two boundaries as observed with schlieren optics. These ratios are ap-
proximately 2.3 : 1. Treatment with EDTA results in nucleic acid-containing
components of 24 and 44 S (fig. 6c) as were likewise observed by schlieren
optics (fig. 5b). The ratio of the amounts of nucleic acid in the two compo-
nents is similar to the ratio of the amounts of material contained in the two
boundaries as observed in schlieren optics, namely, 2:1. The pattern of the
particles incubated in 0.7 M KC1 and observed with absorption optics (fig. 6d)
is, however, markedly different from that observed with schlieren optics (fig.
4£). Components of 75 and 58 S which are observed with schlieren optics are
not observed with absorption optics. In the latter case, however, a new bound-
ary of component 23 S is present to the extent of about 40 per cent of the 43 S
component. The probable explanation of this behavior is that, at the low con-
centration necessary for the employment of ultraviolet optics, the 80 S and the
59 S particles are further dissociated to an undetectable concentration and a new
component of lower sedimentation appears. The 24 S component observed
with ultraviolet optics in potassium chloride-treated preparations is presumably
similar to or identical with the 25 S component observed in solutions treated
with EDTA. Thus, the original particles and their dissociation products all
have similar ratios of ultraviolet-absorbing material to mass.

The intrinsic viscosity of microsomal particles in media which promote dis-
sociation was studied in order to find out what over-all changes in frictional
coefficient are attendant upon dissociation. These experiments were performed
in versene and in potassium chloride solutions at low temperature since at
higher temperature the system in versene aggregates and the system in potas-
sium chloride shows evidence of ribonuclease action. Even though particle
preparations were clarified twice by low-speed centrifugation, the possibility
of the presence of small amounts of aggregates cannot be excluded. The in-
trinsic viscosity of the microsomal particles in phosphate buffer was found to
be 0.11 (100 cc/g) (fig. la), identical with the value found for 24° C. In water,
the value was 0.12/100 cc/g, an increase which may be due to the electroviscous
effect. In solutions of dissociated particles in versene at p¥L 6.8 and 9.0, and in
potassium chloride (fig. lb, c, d), a slightly higher value of reduced viscosity
was obtained at high concentration. The intrinsic viscosity of these solutions,
however, was again found to be 0.11 to 0.12. Thus, no change in intrinsic
viscosity is observed upon dissociation of the particles. The most reasonable
explanation for this result is that the units of 60, 40, and 25 S have the same
frictional coefficient as the 80 S particles.

The above experiments on the dissociation of the 80 S particles can be sum-
marized as follows:

1. When magnesium and calcium are removed from microsomal particles
(79 to 81 S), three ultracentrifugally identifiable components are obtained as

164

MICROSOMAL PARTICLES

E
en

(a)

(b)

8-«

O ,4

~ 12

P04 pH 6.5

.~*~ .10

.16

KCI

0.7 M

.14

o^~- — ""°

^—^-^o

o
o

EDTA

pH 68

(_>

CI)

16
.14
.12

EDTA PH9.o

(c)

m9/cc

12 16

(d)

Fig. 7. Reduced viscosity of particles under dissociating and nondissociating conditions.
(a) In 0.025 fx. P04, pH 6.5, with 1 X 10~4 M MgCU. (£) In 0.7 M KCI, pH 6.5, 0.02 p.
K-P04, 0° C, for 3 hours, (c) In 0.03 M EDTA, pH 6.8, 0.02 /* K-P04, 0° C, for 3 hours.
(</) In 0.05 M EDTA, pH 9.0, 0.01 M Tris buffer, 0° C, for 3 hours.

dissociation products. At infinite dilution these components have sedimenta-
tion coefficients of 59 to 61 S, 40 to 43 S, and 25 to 27 S.

2. All these units contain nucleic acid and probably have RNA/protein ratios
similar to that of the 80 S particles.

3. The mixtures, and perhaps the individual units, of 60, 40, and 25 S com-
ponents have frictional coefficients similar to that of the 80 S particles.

4. Under conditions in which only 60 and 40 S units are formed by dissocia-
tion of 80 S particles, the two components occur in an amount ratio of 2.2-2.3
to 1 respectively in both schlieren and absorption optics. Treatment with EDTA
over a wide range of concentration of both EDTA and particles results in the
formation of only 40 and 25 S units. These are present in an amount ratio
of 2:1, indicating that the two units in the system are in a stable state.

On the basis of the above observations, the molecular weights of the 60, 40,
and 25 S components can be estimated to be 2.6 to 3.0 X 106, 1.3 to 1.5 X 106, and
6.5 to 7.5 X 10\ respectively. The ratios between the molecular weight of the
80 S particles and the molecular weights of the successively smaller units are
then in the series 6:4:2:1. Chao has reached a similar conclusion for the 60 and
40 S components of yeast particles [4|. Reports of Wagman, and of Tissieres
and Watson, in this meeting also tend to support this formulation. Both groups
find that the 40 S particle from Escherichia coli is spherical, with an RNA/pro-
tein ratio close to unity, and a molecular weight of 1.3 to 1.8 million. Since the
40 S particle is spherical, the difference between its sedimentation coefficient and

TS'O 165

the sedimentation coefficients of the 60 and 80 S particles of E. coli reported by
Bolton et al. in this meeting is most probably due to differences in molecular
weight. These would then form the series 3:2:1. In addition, particles of 25 S
have been found as dissociation products of the 40 S particle of E. coli by
Tissieres and Watson, this too having a spherical shape in the electron micro-
scope, as observed by Hall. A 25 S component has also been reported by Chao
[4] as one of the dissociation products of yeast particles.

The following scheme is proposed to account for the experimental findings
concerned with the dissociation of the 80 S particles. When a certain fraction of
the magnesium ion is removed from the system (about 50 per cent as based on
the binding constants of RNA and phosphate [5]), the 80 S particle dissociates
reversibly to form a 40 S unit, one-third of the original particle, and a 60 S unit,
two-thirds of the original particle. When larger amounts of magnesium ions
are removed (over 95 per cent, by versene), not only does dissociation of the
80 S particles to 40 and 60 S units occur, but in addition the 60 S unit also is
degraded, possibly irreversibly, to form a 40 S unit and two 25 S units. The
final result of such a dissociation should be a system containing two 40 S units
and two 25 S units per original 80 S particle. This formulation, then, fits the
experimental findings both as to the mass ratio of 2:1 between 40 and 60 S units
and as to the mass ratio of 2:1 between 40 and 25 S components.

The magnesium and calcium contents of the particle have been analyzed by
flame spectrophotometry." There are 3.0 to 3.2 umoles of magnesium per 12
umoles of RNA-phosphorus or per 10 mg dry weight of particles. All the mag-
nesium appears to be extractable by 0.5 N TCA at 03 C. Assuming that cal-
cium can also be completely extracted by TCA, then, again, per 12 umoles of
RNA-phosphorus, there is 0.45 to 0.55 umole of calcium, about one-sixth of
the amount of magnesium. Thus, the particles contain 3.5 to 3.7 umoles of
magnesium and calcium per 12 umoles of phosphate, or 1 mole of divalent ions
for each 3.3 ±0.2 umoles of phosphate.

It is of interest to estimate the apportionment of these divalent ions between
the RNA and the protein. Combining the data on molecular weight (4.5 X 10c),
RNA content (40 per cent), and content of divalent ions per mole of phos-
phorus, we may calculate that there are 1.5 XlO3 moles of magnesium and cal-
cium per mole of particle. Since the cation binding capacity of the microsomal
protein is unknown, we introduce for comparison the results that may be cal-
culated for bovine serum albumin, a protein with a high proportion of dicar-
boxylic amino acids. According to Carr [6], there are 8 calcium binding sites
per molecule at pW 7.4. The total amount of protein per mole of microsomal
particles is equivalent to 36 moles of bovine serum albumin. This protein
would therefore bind no more than 300 moles of calcium, and would account
for less than 20 per cent of the divalent ions bound by the particles. We con-

2 The technical assistance of Mr. Merck Robison, Carnation Company, is gratefully
acknowledged.

166 MICROSOMAL PARTICLES

elude, therefore, that more than 80 per cent of the divalent ions are bound by
RNA. Experiments with pancreatic ribonuclease further support this con-
clusion. By the action of ribonuclease, a partial separation of protein and
degraded nucleic acid of the particles is achieved. Most of the protein precipi-
tates from solution, leaving behind a mixture of nucleotides and polynucleo-
tides. The distribution of magnesium between supernatant and precipitate
should provide information concerning the binding sites of the magnesium
ions. It was found that, after RNAase had acted on the particles for 2 hours,
85 per cent of the magnesium had been liberated into the supernatant, which
contains 65 per cent of the nucleotide phosphate and only 10 to 15 per cent of
the protein. If we assume that 80 per cent of the magnesium is associated with
RNA in the particle, then half of the phosphate groups in the RNA exist in
the form of magnesium salts.

Attempts to study the protein of the particle have been made with RNAase.
After 3 hours' incubation with 7 ug/ml of pancreatic RNAase in a 1 per cent
solution of particles at 27° C, the solution becomes turbid. Between 85 and
90 per cent of the total protein and 8 per cent of the total phosphorus are sedi-
mentable by low-speed centrifugation, leaving 10 to 15 per cent of the protein
and 90 per cent of the phosphorus in the form of mono- or oligonucleotides in
the supernatant. That the protein aggregation is related to RNAase action and
not to the presence of nucleotides was shown by experiments in which large
amounts of 2,3'-phosphate nucleotides were added to particle preparations with-
out aggregating effect. The protein aggregate is insoluble in buffer at pH 4 to
11, in strong salt solution, in acid, in urea, or in performic acid, but does dissolve
in alkali at pH 12 to 13 as well as in 80 per cent saturated guanidium chlo-
ride at pH 8 to 9. If the guanidium ions are removed by dialysis or if the pH
is lowered to 10 to 11, the protein again precipitates.

The action of RNAase on the particles is enhanced by addition of potassium
chloride or phosphate buffer and is suppressed by addition of magnesium. It
was also found that there is a small amount of contaminating RNAase in our
preparation of particles. Incubation of particles in phosphate buffer (0.05 u,
pH 6.5) for only 2 hours at room temperature hydrolyzes 4 to 5 per cent of the
RNA. This rate can be increased by 8 times by addition of potassium chloride
(0.15 to 0.7 M), and it can be suppressed back to 1.5 to 2 times by addition of
magnesium chloride (0.001 M) with the potassium chloride.

The present studies shed little light on the nature of the binding between
RNA and protein. Electrostatic forces cannot be the only ones involved, since
at least half of the phosphate groups of the RNA are present in the form of
salt linkages with magnesium and calcium. In addition, RNA does not sepa-
rate from protein in the ultracentrifuge in the presence of 0.7 to 1.0 M KCl,
nor can protein be separated from nucleic acid by alcohol precipitation in such
solutions. Agents which denature proteins, such as phenol or detergent, have
been successfully applied to separate RNA from the protein of the particle.
The protein, however, aggregates as the RNA is removed, as shown by the

TS'O 167

experiment with RNAase. These findings, coupled with the fact that urea does
demolish the particle, suggest that hydrogen bonding between RNA and pro-
tein may be an important factor in holding the two constituents together.

Microsomal particles have been isolated from sections of pea seedlings that
have been incubated with C14-leucine for 1 hour.3 The particles, after a series
of centrifugations and dialyses, were shown to be void of contaminating free
C14-leucine, by two methods. The specific activity (cpm/mg protein) of such
particles was the same as that of particles which were washed further by hot
TCA and 1 N NaOH. Moreover, acid hydrolysis of the microsomal protein
after treatment with dinitrofluorobenzene yields over 98 per cent of the C14-
leucine as free leucine, indicating that most of the C14-leucine is linked inside
the protein molecule. With such a preparation of labeled 80 S particles, we
wish to find answers to two specific questions.

The first question is based on the idea that only about 1 to 5 per cent [7] of
the protein in the particle is being actively synthesized in the particles. It has
been suggested that this newly formed protein then passes into the cytoplasm.
In the dissociation studies, it is found that after treatment with EDTA, pH
6.5, most of the nucleoproteins aggregate and sediment out of solution, leaving
about 6 per cent of protein in the supernatant. After the action of RNAase,
there is also about 10 per cent of protein which stays in the solution with the
nucleotides, while 90 per cent of the protein precipitates out of solution. When
the labeled particles were subjected to the above two treatments, only a small
percentage of the total counts of the labeled protein remained in the super-
natant, while over 90 per cent of the counts precipitated with the aggregates.
The supernatant proteins obtained from these two treatments were precipitated
by TCA and were shown to have specific activities no higher than that of the
aggregated nucleoproteins. This experiment suggests that substantial removal
of magnesium and RNA does not liberate a large percentage of labeled proteins
or proteins of very high specific activity from the isolated particles.

The second question is based on the idea that RNA is a template [8]. Thus,
is there a large amount of radioactive amino acids in the particle attached to
RNA through covalent bonds? The RNA was separated from protein by
phenol as well as by density gradient centrifugation in cesium chloride. In-
variably, very few if any counts could be found in the RNA. Furthermore,
the soluble nucleotide fraction of the supernatant after deproteinization by
TCA, obtained by RNAase treatment of the particles, also contained very few
counts. Therefore, if amino acids are attached to RNA through covalent bonds,
the total amount of such material at a given time is likely to be very small.

In summary, microsomal particles from pea seedlings consist of smaller units
of nucleoproteins cemented together through linkage of magnesium ions and
the phosphate group of RNA. RNA and the protein (s) in these units are held

3 Experiments with C14-amino acids were performed in cooperation with Dr. Clifford
Sato.

168

MICROSOMAL PARTICLES

tightly together, probably through hydrogen bonds. Removal of magnesium
ions and hydrolysis of RNA from the particles labeled with C14-amino acids in
the cell did not liberate labeled amino acid or protein (s) of high specific activity
from the particles.

REFERENCES

1. P. O. P. Ts'o, J. Bonner, and J. Vino-
grad, /. Biophys. Biochem. Cytol., 2, 451
(1956).

2. Fu-Chaun Chao and H. K. Schach-
man, Arch. Biochem. Biophys., 61, 220
(1956).

3. M. E. Petermann and M. G. Hamil-
ton, /. Biol. Chem., 224, 725 (1957).

4. Fu-Chaun Chao, Arch. Biochem. Bio-
phys., 70, 426 (1957).

5. J. S. Wieberg and W. F. Neuman,
Arch. Biochem. Biophys., 72, 66 (1957).

6. C. W. Carr, Arch. Biochem. Biophys.,
43, 147 (1953).

7. J. W. Littlefield, E. B. Keller, J. Gross,
and P. C. Zamecnik, /. Biol. Chem., 217,
111 (1955).

8. H. Borsook, /. Cellular Comp. Phy-
siol., 47, Supplement 1, 35 (1956).

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Full text of "Microsomal particles and protein synthesis; papers presented at the First Symposium of the Biophysical Society, at the Massachusetts Institute of Technology, Cambridge, February 5, 6, and 8, 1958"

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